ProcartaPlex™ Human Growth Factor Panel, 11plex - FAQs

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113 product FAQs found

在我的 ProcartaPlex 检测数据分析过程中，我收到一条警告消息，指出存在高度聚集的珠子。我应该怎么办？

以下是此问题的可能原因和解决方案：
o 检查协议设置（确保选择正确的 DD 设置）。
o 检查鞘液液位并清空废液。
o 在获取板之前，在 Luminex 仪器上运行校准和验证珠。

在我的 ProcartaPlex 检测数据分析期间，珠子落在珠子图上珠子区域的下方或左下方。为什么是这样？

以下是可能的原因和问题的解决方案：

•这通常表明微珠已经光漂白。这个问题也可能由于将微珠暴露于有机溶剂中引起。不幸的是，必须重复该检测，因为微珠不能恢复。必须保护珠子免受光和有机溶剂的影响。
•或者，仪器可能在其测量中关闭，或者可能存在校准问题。致电制造商进行服务预约。

在我的 ProcartaPlex 检测数据分析期间，珠子没有出现在门控区域中。发生了什么？

这表示最后一步使用了不正确的缓冲液。必须使用试剂盒中提供的洗涤液洗涤微珠并在微珠上样至Luminex仪器之前将其重悬。溶液的渗透压将影响微珠的尺寸，而微珠尺寸的任何变化都将改变仪器的检测。

我所有的 ProcartaPlex 检测孔的珠数都不稳定。什么地方出了错？

以下是一些建议：
o 在获取板之前，在 Luminex 仪器上运行校准和验证珠。
o 查看仪器设置并确保它们适合正在运行的检测（调整针高，确保选择正确的珠门和正确的 DD 设置）。
o 在仪器上采集前摇动板以重新悬浮珠子。
o 将珠子涡旋 30 秒，然后再将它们添加到板中。
o 清洗：在清空板之前，不要忘记将板放在手持式磁性洗板机上约 2 分钟。

我的 ProcartaPlex 检测标准曲线的微珠计数正确且规则，但对于我的样品，微珠计数不稳定，我收到一条警告消息，提示“采集中至少有一个区域未达到最大计数”。我应该怎么办？

这种模式表明样本基质效应。以下是一些建议：
o 确认样品已澄清且不含碎片和脂质（建议离心 5-10 分钟）。
o 确认血清、血浆样品的样品与测定稀释剂的比例至少为 1:1。对于细胞裂解物或组织匀浆，确认样品已在测定缓冲液中适当稀释，以将裂解缓冲液中的去污剂浓度降低至≤0.01%。对于其他样品类型，可能需要进一步的样品优化。

我的 ProcartaPlex 检测样品的读数表明它们低于可检测限度。你有什么建议？

以下是一些建议：
o 您感兴趣的蛋白质水平可能低于检测的检测限。高灵敏度多重试剂盒可用于大多数细胞因子。
o 验证您的标准曲线（寻找平台、异常曲线拟合、异常值）。

我的 ProcartaPlex 检测样品的读数表明它们高于可检测限度。我应该怎么办？

以下是一些建议：
o 可能需要样品优化：用合适的稀释剂稀释样品并重新运行。
o 验证您的标准曲线（寻找平台、异常曲线拟合、异常值）。

如何处理用于Luminex检测平台的细胞裂解物（提取细胞蛋白质）？

下述方案已经应用于多种人类和小鼠细胞系。您应该为您自己的应用优化细胞提取操作。

细胞裂解操作

非贴壁细胞：
低速离心以沉淀细胞。从沉淀中移去培养基，用冰冷的PBS洗涤两次。去除PBS，将细胞沉淀重悬于细胞裂解缓冲液中（推荐细胞裂解液浓度为2-5 mg/mL）。在冰上孵育15分钟，偶尔涡旋。将裂解物转移至微量离心管中，并在2-8℃下以14000rpm离心10分钟。 将澄清的裂解液等分至干净的微量离心管中，并测定总蛋白浓度。

贴壁细胞：
从细胞中去除培养基，用冰冷的PBS洗涤两次。去除PBS，加入细胞裂解缓冲液（推荐细胞裂解液浓度为2-5μg/mL），并在冰上孵育15分钟。收集细胞裂解液，并将其转移至微量离心管中。在2-8℃下以14000rpm离心10分钟。 将澄清的裂解液等分至干净的微量离心管中，并测定总蛋白浓度。裂解液应在-80°C下冷冻储存，或在收集后立即进行分析。冷冻样品避免多次反复冻融。分析前，将样品完全解冻、充分混合并通过离心（14000rpm，10分钟）来澄清，以防止堵塞过滤板。

推荐的细胞裂解缓冲液：

NP40细胞裂解缓冲液（货号FNN0021）
注意：使用NP40裂解缓冲液制备的裂解液必须在检测之前稀释至少5倍。
或者使用下述配方进行配置：50 mM Tris，pH 7.4，50 mM NaF，260 mM NaCl，1 mM Na3VO4，5 mM EDTA，0.02% NaN3，1% Nonidet P40

NP40细胞裂解缓冲液（不含蛋白酶抑制剂混合物和PMSF）在2-8°C下的稳定期为2-3周，或在-20°C下分装保存6个月。

使用前在NP40细胞裂解缓冲液中新鲜添加：
•1 mM PMSF（DMSO中的0.3 M原液）
•蛋白酶抑制剂混合物（Sigma，货号P-2714）

替代细胞提取缓冲液

细胞提取缓冲液（货号FNN0011）
注意：使用细胞提取缓冲液制备的裂解液必须在检测之前稀释至少10倍。

或者

10 mM Tris，pH 7.4
2 mM Na3VO4
100 mM NaCl
1% Triton X-100
1 mM EDTA
10% 甘油
1 mM EGTA
0.1% SDS
1 mM NaF
0.5% 脱氧胆酸盐
20 mM Na4P2O7

细胞提取缓冲液（不含蛋白酶抑制剂混合物和PMSF）在2-8°C下的稳定期为2-3周，或在-20°C下等分保存6个月。

使用前在细胞提取缓冲液中新鲜添加：
•1 mM PMSF（DMSO中的0.3 M原液）
•蛋白酶抑制剂混合物（Sigma，货号P-2714）

如何处理用于Luminex检测平台 的唾液样品？

我们没有在内部专门测试唾液样本，因此这些说明只是我们的建议。唾液含有几种蛋白水解酶。将样品离心并确保不要将任何细胞材料或碎片移入测定板是很重要的。我们建议在样品中添加一些抗蛋白酶（例如，trasylol 或 aprotinine，10 至 50 U/mL）以保护蛋白质免受酶降解。然后，您可以在 4 摄氏度以 1000 x g 的速度将样品离心 10 分钟以去除颗粒。立即使用或在 -80 摄氏度下储存等分试样。避免多次冻融循环。 

如何处理用于Luminex检测的口腔粘膜渗出液样本？

隔离牙齿周围的部位，将一张牙周滤纸插入牙齿周围的牙龈袋中，持续30秒。取出滤纸，在室温下使用50μL PBS提取4次，每次5分钟。每次提取物可以组合和单独进行分析。在应用于检测之前，用检测稀释液稀释2倍。

如何处理在Luminex检测中使用的宫颈分泌物样品？

收集后，应立即将宫颈取样海绵放置于冰上。样品应在-20°C下储存长达一周，然后储存于-80°C直至需要检测准备。解冻后，应将海绵称重并放入Eppendorf管中，每次转移均要使用乙醇处理过的镊子进行。向每个管中加入200μL冰冷的提取缓冲液（配方见下文），并在4℃下孵育过夜。 然后将海绵和提取缓冲液转移到具有0.2μm醋酸纤维素过滤器的微量离心管中，并在4℃下以13000rpm离心10分钟。 然后测试洗脱液中的细胞因子表达。

提取缓冲液

50 mM HEPES，pH 7.5
1 mM Na3VO4
150 mM NaCl
1 mM NaF
1 mM EDTA
0.1% Tween 20
25 mM EGTA
10% 甘油

如何处理在Luminex检测平台上使用的支气管肺泡灌洗（BAL）液样品？

支气管肺泡灌洗（BAL）液应收集于无菌注射器中，并在分析之前持续保存于冰上。或者，可以将BAL等分至需用样品大小并冷冻（以便只会经历一次冻融）。分析前，所有样品需要通过离心（14000rpm，10分钟）和/或过滤来澄清，以防止堵塞过滤板。在应用于检测板之前，用检测稀释液稀释2倍。

如何处理在Luminex检测平台上使用的脑脊液（CSF）？

分析前，所有样品需要通过离心（14000rpm，10分钟）和/或过滤来澄清，以防止堵塞过滤板。然而，CSF具有相对较低的粘度，并且除非存在感染状态（WBC的丰度），否则其不需要澄清并可以直接应用于检测。使用神经科学缓冲液试剂盒（货号LNB0001）中提供的检测稀释液稀释2倍。 

如何处理在Luminex检测平台上使用的关节液（synovial fluid）样本？

将关节液收集到非肝素化管中，并在样品采集的30分钟内以1000×g离心10分钟。在进行随后的分析之前，关节液的无细胞部分应储存在-80℃。分析前，所有样品需要通过离心（14000rpm，10分钟）和/或过滤来澄清，以防止堵塞过滤板。在加入检测之前，用检测稀释液以1：1稀释样品。参考文献：Raza K等人（2005） 关节炎研究与治疗7（4）: R784–R795。

如何处理在Luminex检测平台上使用的组织匀浆样本？

我们建议遵循下面提供的的方案，这是基于组织提取试剂I（货号FNN0071）开发并显示了ELISA和Luminex技术之间的良好相关性。该方法已应用于多种组织类型。但是，我们建议您针对所使用的每种组织样品进行优化。可以使用类似的提取试剂/裂解缓冲液。

1.在使用前将蛋白酶抑制剂添加到组织提取试剂中。
2.称量组织样品。
3.每1克组织加入10 mL组织提取试剂。
4.匀化组织。
5.以10000rpm离心样品5分钟以沉淀组织碎屑。
6.收集上清液。按照试剂盒中提供的操作步骤进行适当的稀释；这是为了防止/最小化提取液或裂解缓冲液中存在的去垢剂对抗体 - 抗原结合的潜在抑制。通常，组织匀浆或细胞裂解物（取决于所用的裂解缓冲液）需要稀释5至10倍，以将去垢剂浓度降低至≤0.01％。然而，基于目标细胞因子/蛋白质的浓度，不同的试剂盒/样品可能需要用检测稀释液或标准品稀释液进一步稀释。

注意：如果要储存样品，请将其等分并冷冻于-80°C。避免多次反复冻融。

如何处理在Luminex检测平台上使用的细胞培养上清液样本？

细胞应处于对数生长期。在适当的细胞培养瓶中根据需要刺激细胞。使用无菌技术，用移液器吸移所需体积的条件细胞培养基，并将培养基转移到干净的聚丙烯微量离心管中。在冷冻微量离心机中，4℃ 14000rpm将培养基离心10分钟，以去除细胞或细胞碎屑。将澄清的培养基等分至干净的聚丙烯微量离心管。这些样品即可用于检测。或者，可将澄清的培养基样品等分并储存在-80℃用于将来分析。避免多次反复冻融。将冷冻样品置于冰上使其解冻，并应在解冻后立即进行测试。分析前，解冻的样品应立即在冷冻微量离心机中通过离心（4℃ 14000rpm离心10分钟）澄清，以防止堵塞Luminex探针和/或过滤板。遵循试剂盒提供的检测程序进行适当的稀释。

如何处理在Luminex检测平台上使用的血浆样品？

在冷冻离心机中以2000×g离心10分钟，将细胞从血浆样品中分离。需要在该力下离心以去除样品中的血小板。用无菌巴斯德移液器将上清液转移到冷却的干净聚丙烯管中。处理时使样品保持在2-8°C。

如果血浆有待日后分析，请将其分装至聚丙烯微量离心管并储存在-80℃。 避免多次反复冻融。分析之前，将样品置于冰上，使其解冻。所有血浆样品应在分析前在冷冻微量离心机中通过离心（4℃下以14000rpm转速离心10分钟）澄清。遵循试剂盒提供的检测程序进行适当的稀释。 

如何处理在Luminex检测平台上使用的血清样品？

应使用无热原/无内毒素的试管采集血清样品。将全血在室温下静置15-30分钟，使其凝固。在4℃冷冻离心机中以1000-2000×g离心10分钟以分离细胞。用无菌巴斯德移液器将上清液转移到冷却的干净聚丙烯管中。处理时使样品保持在2-8°C。 

如果血清有待日后分析，请将其以0.5 mL体积分装后储存在-80℃。 避免多次反复冻融。如果可能，避免使用溶血或有脂血症的血清。我们建议，分析前，在解冻后立即通过离心（14000rpm，10分钟）和/或过滤来澄清样品，以防止堵塞过滤板和/或探针。按照试剂盒提供的检测程序进行适当的稀释。

ProcartaPlex 多重检测对每种分析物的结果是否与我目前的 ELISA 检测相同？

ProcartaPlex 多重检测基于 Luminex xMAP 技术，提供了一个多功能平台，为用户提供了更大的灵活性和更多的分析物检测选项。无论您是测试单一分析物还是多种分析物，ProcartaPlex 多重分析都使用高效、易于遵循的方案提供准确的分析性能。这些检测中的每一个都经历了我们为 ELISA 进行的相同的开发、验证、制造和质量控制标准化。每批 ProcartaPlex 多重检测和 ELISA 检测都经过适当的样本类型（即物种特异性血清、血浆和细胞培养上清液）的完全验证，并且每批都根据以下性能特征进行评估：
o特异性 - 筛选每个分析物以确保在多重测试中与其他分析物没有明显的交叉反应
o灵敏度 - 对每个分析物的功能灵敏度（与背景的区别）和检测下限 (LLOD) 进行评估
o精密/准确度多重检测具有良好的检测内精密度 (<10% CV)、检测间精密度 (<10% CV) 和批次间一致性 (<20% CV)；这些值与大多数 ELISA 测试相当或更好

ProcartaPlex 多重检测定期针对匹配的 ELISA 进行测试。因此，您可以轻松地从 ProcartaPlex 检测切换到 ELISA，反之亦然，结果可靠。我们的大多数 ProcartaPlex 检测使用与我们传统的基于平板的 ELISA 相同的抗体对，从而导致两种检测之间的高度相关性 (R2 > 0.9)。

Luminex xMAP技术如技术如何实现在一个孔中同时检测多个分析物？

Luminex xMAP技术基于聚苯乙烯或顺磁微球或微珠，其内部用不同强度的红色和红外荧光团标记。每个带颜色的微珠都有一个唯一的编号，称为“微珠区域”，以区分不同的微珠。对于Invitrogen多重免疫检测试剂盒，每个微珠组用一种捕获抗体包被以用于一种特定分析物的检测。然后可将多种分析物特异性微珠在96孔检测板的单个孔中混合，使用其中一种Luminex仪器同时进行多种靶标的检测和定量。我们提供使用聚苯乙烯珠或顺磁磁珠进行的多重检测试剂盒。观看视频(https://www.youtube.com/watch?v=kEdLGcGXrs4&feature=youtu.be)，了解如何在Luminex仪器平台上使用Invitrogen多重微珠试剂盒同时检测多种蛋白质。

什么是Luminex检测平台？

具有确定光谱特性的微珠与蛋白质特异性捕获抗体偶联，并与样品（包括已知蛋白质浓度的标准品、对照样品和测试样品）一起添加到微孔板的孔中。靶蛋白在2小时的孵育过程中与捕获抗体结合。洗涤微珠后，加入蛋白质特异性生物素化的检测抗体，并与微珠一起孵育1小时。然后，去除过量的生物素化检测抗体，加入偶联有藻红蛋白的链霉亲和素，并孵育30分钟。 SAV-RPE与生物素化的检测抗体结合，形成四元固相夹心。洗涤去除未结合的SAV-RPE后，用Luminex检测系统分析微珠。通过监测微珠的光谱特性和相关的藻红蛋白（RPE）荧光的量，可以测定一种或多种蛋白质的浓度。Luminex技术与以下Luminex分析仪兼容：

•MAGPIX系统 — 经济实惠、高效、紧凑设计
•Luminex100/200系统 — 多功能、高效、广泛用于多重检测
•FLEXMAP 3D系统 — 高通量（同时分析高达500个）和自动化兼容

What is the size of the Luminex beads you currently use?

The beads used in our Luminex instrument-compatible ProcartaPlex and QuantiGene Plex assays are 6.5 micron superparamagnetic beads.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I am interested in performing Luminex assays using BioSource kits, and I have a Luminex xMAP system. Besides the kits and system, what other reagents and equipment will I need?

The following is a list of general lab supplies that are required for running BioSource immunoassays on the Luminex xMAP system:
1) Sonicating water bath
2) Orbital shaker
3) Vortexer
4) Repeating and/or multi-channel pipetter (not required, but recommended)
5) Calibrated adjustable precision pipettes, with disposable plastic tips
6) Glass/plastic tubes and racks for preparing reagents
7) Graduated cylinder and container for preparing wash solution
8) Aluminum foil
9) Deionized or distilled water.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Do the Luminex beads require special care in handling?

The Luminex beads should be protected from light because they are susceptible to photobleaching. We recommend protecting the beads by keeping containers covered with aluminum foil during all incubation steps, and exercising care during handling. The beads should not be frozen, subjected to excessive heat, or exposed to organic solvents.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Why would the Luminex acquisition software display "Sample Empty" messages during analysis?

(1) The user did not properly aliquot the diluted beads, such that no beads were actually added to the wells (make sure that the bead concentrates are sonicated and vortexed well, then check the pipet tip to ensure that air bubbles were not drawn up)
(2) The user missed loading diluted beads to some wells, which is likely since the small volume is clear and difficult to visualize in the clear plastic plate (we have now addressed this customer difficulty by coloring each of the Buffer Reagent Kit components)
(3) The user applied too much vacuum pressure at some point during the wash steps, or allowed the pressure to spike even once, such that the filter membrane tore in a few wells releasing the beads (make sure that the vacuum manifold pressure is kept below 5mm/in Hg, depending on their system -- a good rule of thumb is that it should take a full 3-second count to GENTLY empty the wells of 200uL)
(4) The user did not properly sonicate and vortex the beads prior to dilution, such that the percent of bead aggregation was high and the instrument was unable to find enough single beads to meet the events/bead value designated by the customer (make sure that the Bead Concentrate tube is put into the waterbath all the way to the cap, since the tube is hollow until the top third)
(5) The user lost beads by shaking the plate too aggressively or handling it improperly (make sure that the orbital shaker is set to a speed that allows for maximum vortex in the wells without spillage)
(6) The user exposed the beads to an excess of light during storage or running of the assay, such that some but not all of the beads were photobleached and therefore falling outside the acceptable range for each bead region (make sure that the plate is covered on the top/sides with foil throughout the assay, away from Windows and spotlights, and that the bead component of the kits is stored in the dark)*
(7) There was a clog in the sample needle, such that the instrument was unable to take up enough sample to meet the number of events requested per bead region (suggest that the user follow the manual instructions for dislodging a clog, which include several Back Flush steps and may require removal of the needle for sonication with probe alignment).

* Some of the older Antibody Bead Kits still have clear plastic tops instead of black ones. In cases where customers store kits in lit refrigerators, or keep them open on the lab bench, even a few hours of light exposure is enough to photobleach beads. It is important to note, in general, that higher number bead regions are more susceptible to photobleaching. In order to draw conclusions about the source of the difficulty, we would ask to see the data, specifically the Masterplex QT file, which would enable us to examine the pattern of "Sample Empty" occurrences in addition to the bead counts per well.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What are the Luminex beads made of?

The beads are made of polystyrene.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How do the Luminex 200 Instrument System, Luminex FLEXMAP3D Instrument System and Luminex xMAP INTELLIFLEX System simultaneously monitor concentrations of several different analytes in a single sample?

Each protein detected requires a different bead region to differentiate between analytes. The Luminex 200 Instrument System, Luminex FLEXMAP3D Instrument System and Luminex xMAP INTELLIFLEX System are outfitted with two lasers. The first laser is a red diode laser that classifies beads (region) based on color and hence identifies which protein is being measured. The second laser is a green YAG laser that quantitates reporter fluorophore (R-Phycoerythin, RPE) associated with the bead surface and determines the amount of the protein that is bound to the bead. The beads pass through the system at a rate of several thousand per second, excited first by the red laser and then the green laser. The results obtained show protein identity and the intensity of the signal, respectively. The intensity of the signal is directly proportional to the concentration of the analyte captured in the sample. Therefore, by monitoring the spectral properties of the beads and the amount of associated RPE fluorescence, the concentration of one or more proteins can be determined.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Do you offer a Mac-compatible version of the ProcartaPlex Analyst Software?

Unfortunately, we do not currently offer a Mac-compatible version of the ProcartaPlex Analyst Software. ProcartaPlex Analyst Software is compatible with PC, or xPONENT Software is available on Luminex instruments to analyze your data.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Which Luminex instruments are available for multiplex detection and analysis?

Here (https://www.thermofisher.com/us/en/home/life-science/antibodies/immunoassays/procartaplex-assays-luminex/luminex-instruments.html) is an overview of Luminex instruments we offer for multiplex detection and analysis.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I am getting poor recovery with my ProcartaPlex multiplex immunoassay. What can I do?

Here are possible causes and solutions:
- The wrong cell culture media may have been used to prepare the standards. We recommend using the same cell culture media that was used to culture the cells.
- The samples and antigen standards may not have been stored on ice. We recommend preparing the samples and standards on ice before setting up the assay.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I am seeing low signal or sensitivity in my ProcartaPlex multiplex immunoassay. What should I do?

Standards may not have been reconstituted and diluted correctly. We recommend preparing fresh antigen standards following the instructions provided in the corresponding ProcartaPlex user guide.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

With my ProcartaPlex multiplex immunoassay, I am getting a low bead count. What is causing this?

Here are possible causes and solutions:
- Volume of the bead solution is too low. We recommend adding 120 µL Reading Buffer into each well and shaking at 600 rpm for 5 mins at room temperature to resuspend beads before reading on the Luminex instrument.
- High bead aggregation. We recommend vortexing the bead suspension well before using in the assay and ensuring that the beads are properly mixed during the incubation steps.
- Dyes contained in the beads are photo-bleached from overexposure to light. We recommend storing the bead solution and the 96-well plate in the dark.
- Samples causing the instrument to clog. We recommend removing the 96-well Flat Bottom Plate and performing a wash and rinse to the instrument and then re-running the assay with further dilution of samples.
- Probe height is incorrect. We recommend referring to the Luminex manual for proper adjustment of the needle height.
- The instrument needle is partially clogged. We recommend replacing or cleaning the needle according to the manufacturer's recommendations.
- Beads stuck to the bottom of the plate. We recommend confirming that the plate shaker is set to 600 rpm and shaking for at least 5 mins before reading.
- Air bubble in the sample loop. We recommend referring to the specific Luminex manual for your assay for proper removal of the air bubble.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

In ProcartaPlex multiplex immunoassays, we are seeing a limited dynamic range for BioPlex software users. Why is this?

The instrument may have been calibrated at high PMT settings. We recommend calibrating the instrument using the CAL2 Low RP1 target value.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I am seeing high CVs with my ProcartaPlex multiplex immunoassay. Why is this?

Here are possible causes and solutions:
- Samples and antigen standards not stored on ice. We recommend preparing the samples and standards on ice before setting up the assay.
- Contamination from re-using the Plate Seal. We recommend using a new Plate Seal for each incubation step.
- Incomplete washing. After adding the standards and samples, it is very important that any excess standards are removed during the wash step.
- Contamination from contents from adjacent wells. Avoid splashing the Wash Buffer during wash steps into adjacent wells.
- Poor pipetting techniques. We recommend using a multichannel pipettor and careful pipette techniques. Avoid touching pipette tips to sides of the wells when adding Wash Buffer.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

With my ProcartaPlex multiplex immunoassay, I am getting a low flow rate. What should I do?

Samples/beads could have been stuck in the flow cell. We recommend removing the 96-well plate and performing a wash and rinse cycle.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Do you offer FlowCytomix assays?

Sorry, the FlowCytomix product line has been discontinued. However, we offer a new antibody and magnetic bead-based detection system, i.e., ProcartaPlex assays for multiplex protein quantitation using the Luminex instrument platform.

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How long does it take to acquire ProcartaPlex assay data on the different Luminex instruments?

With everything working smoothly, here is the approximate read time for one plate on different Luminex instruments:

- FLEXMAP 3D Systems and INTELLIFLEX Systems will read 96 well plates in ~20 min, and 384 well plates in ~75 min.
- Luminex 100/200 Systems will read 96 well plates in ~40 min.
- MAGPIX System will read 96 well plates in ~50-60 min. Please note that these are estimates for the read times of the plates only, and do not include time for calibration or set-up of the instrument.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Is the sensitivity value for a Multiplex ProcartaPlex assay different when compared to the sensitivity for those same analytes assayed as Simplex assays?

In general, sensitivity between Simplex and Multiplex ProcartaPlex assay kits is comparable.

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Why does the ProcartaPlex ULOQ (Upper Limit of Quantitation) and LLOQ (Lower Limit of Quantitation) change between different lots of standards?

We supply the standard protein in a lyophilized form. Lyophilization is a dehydration process that preserves the perishable standard. We have demonstrated that often, the amount of starting protein prior to lyophilization and that of the final product after lyophilization varies (this could be different from analyte to analyte). To ensure that we deliver the most quantitative and reproducible product, we always calibrate the standard after lyophilization and therefore, the concentration range can vary from lot to lot of the standard.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can the ProcartaPlex beads be frozen?

No. Storage of the beads at temperatures below 0 degrees C will damage the beads and render them unusable.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What is the minimum bead count used for statistical significance/analysis in ProcartaPlex assays?

We validate and release our ProcartaPlex assays with a minimum bead count of 50. In our experience, the assays also work with a bead count of 20 though we have not validated them at this bead count.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Is ProcartaPlex assay sensitivity information based on data using the 2 hr or overnight incubation with samples?

The sensitivity information is based on the 2 hr incubation with samples.

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How can I minimize inter-assay variation for ProcartaPlex assays?

To minimize inter-assay coefficient of variation between two independent assay plates, it is very important that the same operator performs the assays. Also, we recommend that you follow the incubation times exactly as they are stated in the manual in order to get consistent results. Other tips for minimizing inter-assay coefficient of variation include performing a standard curve in duplicate on each plate and using the same kit lot.

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I am trying to find out whether the medium/serum used to culture my cells will interfere with the ProcartaPlex assay. Which cell culture media have you tested in-house with ProcartaPlex assays?

In-house, we test ProcartaPlex assays with RPMI + 0.5% FCS. You would have to empirically determine if your specific medium will interfere with the assay.

Note: Our customers have successfully used media containing up to 5% FBS with ProcartaPlex assays. As bovine serum contains TGF beta, the use of FBS or FCS should be avoided with TGF beta ProcartaPlex assays.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I am a Bio-Plex user. How can I generate and export CSV raw data to be analyzed using the ProcartaPlex Analysis App?

Open the Bio-Plex Manager and click on “File”, then “Document Export Properties”, then “Output CSV Format” and set “Analytes Labels” to “Name (Region)”. Choose the desired file “Destination”, and click “OK”. Click “Document Export”, then “Export”, and import the generated .CSV file into ProcartaPlex Analysis App on Thermo Fisher Connect: https://apps.thermofisher.com/apps/procartaplex

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How do I process tear samples for use in a Luminex assay?

We have not specifically tested ProcartaPlex assays with tear samples in-house. However, here is a reference from the literature describing the use of ProcartaPlex assays with tear samples (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161422/pdf/pone.0107370.pdf).

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How do I process breast milk samples for use in a Luminex assay?

We have tested breast milk samples in-house using the serum ProcartaPlex protocol and found no general interference with the breast milk matrix. Please note that we have only tested a few analytes (TNFa, IL-10, TNFR), however, there is no obvious reason why other analytes should not work as well.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What is the size of beads used in the ProcartaPlex assays?

The size of the beads in our ProcartaPlex assays is 6.5 µm.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What data analysis software should I use to analyze the data obtained with my ProcartaPlex assay?

All Luminex instruments come with xPonent data analysis software that can be used. Alternatively, we offer a free and robust data analysis software package, that can be downloaded from our website (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-analyst-software.html).

Additionally, please refer to our ProcartaPlex Assays Support Center (https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/antibodies-immunoassays-support-center/luminex-assays-support.html) for data analysis tips and tricks.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My ProcartaPlex assay data lists some targets as OOR<, what does this mean?

This means that the data obtained in that run are below the detectable limit of the assay. Here are some possibilities and suggestions:
1. Qualify your standard curve using the data analysis tools found in our Luminex Assays Support Center (https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/antibodies-immunoassays-support-center/luminex-assays-support.html).
2. Make sure that your dilution factors are set correctly.
3. It is possible that the functional sensitivity of the standard curve can be extended to better distinguish the data at the lower end of the curve. This can be done by adding one or two further dilutions to the curve and re-running the assay with the samples of lower concentrations.
4. It is possible that the levels of your protein of interest fall below the detection limits of the assay.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Where can I find a complete list of targets for your entire ProtcartaPlex assay portfolio?

A complete list of available targets for our entire ProtcartaPlex assay portfolio can be found on our website by using the ProcartaPlex Panel Configurator (https://www.thermofisher.com/order/luminex/) and choosing the species of interest. Alternatively, this information can be found on our ProcartaPlex Assays page here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-assays.html

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Where can I find a complete list of targets for your human ProcartaPlex assays?

A complete list of available targets for our entire ProtcartaPlex assay portfolio can be found on our website by using the ProcartaPlex Panel Configurator (https://www.thermofisher.com/order/luminex/) and choosing the species of interest. Alternatively, a list of all human ProcartaPlex kits can be pulled up on our website using the search term 'ProcartaPlex Assay Human', or by opening the drop-down menus for Human ProcartaPlex Panels on our ProcartaPlex Assays page (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-assays.html).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My ProcartaPlex assay custom panel selection says: "TGF-beta1 has to be acid treated for proper detection of bioactive form. Recommendation: single plex assay. Alternative: LAP (TGF-beta1)" What is the difference between TGF-beta1 and LAP (TGF-beta1)?

The TGF-beta1 assay requires an acid pre-treatment of the sample to reveal the TGF-beta1 protein and therefore, the assay cannot be combined with other assays. The acid pre-treatment of the sample will destroy the other protein epitopes. However the LAP TGF-beta1 assay does not require an acid treatment, but it will measure only the LAP-TGFbeta1 complex. Neither purified LAP nor purified TGF-beta1 alone can be measured in this particular assay.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My ProcartaPlex assay custom panel selection has notes regarding different dilution factor needed for some of my targets, can these targets still be combined into one panel?

This will depend on your sample type. Often serum and plasma may require dilutions for certain targets whereas culture supernatant does not. However, it is optimal to combine only those targets that require the same dilution.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My ProcartaPlex assay custom panel selection shows bead region conflicts, can my targets still be combined into one panel?

Yes, our Mix & Match team can adjust the bead regions as necessary to accommodate targets in your panel and accommodate instrument parameters (i.e., MagPix can only read 50 bead regions).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I am planning to use the ProcartaPlex Panel Configurator to order a custom panel. How do I know if my list of targets can be multiplexed in a ProcartaPlex assay?

When using the ProcartaPlex Panel Configurator (https://www.thermofisher.com/order/luminex/), your targets will be listed along with any 'notes' or 'cautions' pertaining to your panel selection and sample type.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I have a list of targets that do not already come as a pre-made multiplex ProcartaPlex assay. Can this be custom ordered?

Yes, please use our ProcartaPlex Panel Configurator (https://www.thermofisher.com/order/luminex/) to request a quote for your Mix & Match panel.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I want to run half a plate at a time for my ProcartaPlex assay, can I use the same plate again? Can I purchase more plates?

Yes, you can use half a plate at a time, but make sure to seal the unused half with plate sealing tape to prevent any contamination during the assay. Alternatively, you can purchase extra plates (Cat. No. EPX-88182-000).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I purchase more of the Standard Mix for my ProcartaPlex assay?

This may be possible as a custom order. Please reach out to your Sales Representative or to Technical Support (techsupport@thermofisher.com) with your kit number and lot number, they can work with our manufacturing team to determine availability of lot-specific standards.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Which hand-held magnetic separator should I use with your ProcartaPlex assay kits?

Our ProcartaPlex assays have been validated with the Hand-Held Magnetic Plate Washer (Cat. No. EPX-55555-000). We also recommend the Magnetic 96-Well Separator (Cat. No. A14179) as it has a stable magnetic field distributing beads along the bottom of the wells for maximum retention of beads during wash steps.

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Can I use automated plate washers with your ProcartaPlex assays?

If you have a magnetic automated plate washer, this can be used with our magnetic bead kits.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I need more ProcartaPlex Streptavidin-PE for my assay. Can I purchase this item separately?

Yes, ProcartaPlex Streptavidin-PE (Cat. No. EPX-SAPE-000) is available as a stand-alone item. We offer most of our PorcartaPlex buffers and reagents as stand-alone items. A complete list of what is currently available can be found on our website here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-supplemental-products.html.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I have spilled my ProcartaPlex assay buffer, can I purchase more?

Yes, our Universal Assay Buffer (Cat. No. EPX-11110-000) can be bought separately. We offer most of our ProcartaPlex buffers and reagents as stand-alone items. A complete list of what is currently available can be found on our website here, under "Accessories": https://www.thermofisher.com/us/en/home/life-science/antibodies/immunoassays/procartaplex-assays-luminex/procartaplex-immunoassays/procartaplex-preconfigured-panels.html.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How long does a typical ProcartaPlex assay take to set up and run?

Typical timeline for sample to results for our ProcartaPlex assays is 4.5 hrs.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Do you have any references for your ProcartaPlex assays?

We do list many references for our ProcartaPlex portfolio on our website here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-immunoassay-publications.html. More recent publications can be found using internet search tools such as Google Scholar or similar.

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What species of non-human primates can be used with your NHP ProcartaPlex assays?

We have listed all the species we have tested in our Cross-reactivity Chart for NHP ProcartaPlex Simplex Kits. This chart can be found here (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays/procartaplex-immunoassays/procartaplex-assays.html) by clicking on the link for Non-human Primate ProcartaPlex Panels.

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What is a ProcartaPlex High Sensitivity assay?

ProcartaPlex High Sensitivity assays are designed to measure small concentration differences in cell culture supernatants, plasma, and serum samples with 10-fold lower LLOQs (Lower Limit of Quantification) and sensitivities for all analytes in the femtogram range. They have been further optimized to overcome the sensitivity detection limits of conventional multiplex immunoassays.

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What is the difference between the "Convenience" and the "Combinable" multiplex panels? Can they be multiplexed with each other?

“Convenience” multiplex panels are pre-mixed and optimized bead sets for use without modification. “Combinable” multiplex panels are target sets which can be combined or supplemented by addition of beads from ProcartaPlex Simplex kits. These two types of panels cannot be multiplexed together.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I have more than 5 mixes of standards to combine for my ProcartaPlex multiplex assay, how do I do this?

Different antigen standard set vials can be reconstituted simultaneously as long as the volume of sample type-specific standard buffer is at least 50 µL per vial and equals 250 µL in total volume. Please refer to our application note: Preparation of Antigen Standards (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017832_PrepAntigenStd_Procartaplex_PI.pdf).

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How do I set up the Luminex instrument?

Please refer to the assay protocol for kit-specific instructions and your Luminex instrument user guide for instrument-specific instructions. Prior to running the assay, please ensure that the probe height has been calibrated with the 96-Well Flat Bottom Plate supplied with the kit. Failure to adjust the probe height can cause damage to the instrument or low bead count. The Luminex system allows for calibration of low and high RP1 target values. We recommend RP1 low target value settings for immunoassays. Please refer to the lot-specific Certificate of Analysis provided with the kit for bead region and analyte associations when entering the information into the Luminex Acquisition Software.

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I am planning to run tissue lysates for use on a Procartaplex assay. Can you please provide the protocol?

Please follow the instructions here (https://tools.thermofisher.com/content/sfs/manuals/Preparation-of-Tissue-Homogenate.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My samples have been fixed with paraformaldehyde and processed to separate the serum/plasma fraction after fixation. Are they compatible with ProcartaPlex assays and how should I process them?

We have not tested the use of fixed samples with the ProcartaPlex assay and therefore cannot confirm compatibility at this time.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

I have bought several ProcartaPlex Simplex Bead Sets. Which premixed standard do I have to use in my assay?

Please refer to the lot-specific Certificate of Analysis to check the source of the Standard Mix (i.e., Standard Mix A, B, or C) included with each kit. When combining Simplex Bead Sets with other Simplex Bead Sets or Multiplex kits, multiple vials of the same premixed standard (Standard Mix A, B, or C) may be shipped. If the combination of analytes you want to analyze in your multiplex assay includes two analytes that require the same premixed standard, use only one vial of the premixed standards to prepare the standard curve. If the kits you want to combine include different lots of the same premixed standard, please choose one vial (of any lot) for your multiplex assay.

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How many standard points do we need to prepare when using the ProcartaPlex kits?

You will need to prepare a 7-point standard curve as well as a blank.

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Can a finished ProcartaPlex plate be read multiple times on the Luminex instrument without a significant signal loss?

Yes, it is possible to reread a finished ProcartaPlex plate without a loss in the signal or the number of beads counted. Please note that the Luminex instrument adds additional liquid to the wells with each analysis. It is possible that the wells may become overfilled with fluid after the third analysis. Hence, we do not recommend reading the ProcartaPlex plates more than two times.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I combine ProcartaPlex kits, which were not purchased on the same order? For example: I purchased a kit 3 months ago and another kit today. Can I combine them or do the kits have to be shipped on the same order?

Yes, you can combine ProcartaPlex Combinable panels and Simplex kits from different orders to run a bigger multiplex experiment. You will want to ensure that there is no overlap of bead regions between the kits you are combining. Please refer to the online ProcartaPlex Panel Configurator (https://www.thermofisher.com/order/luminex).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Do I need to purchase a Basic Kit when combining Simplex kits with a Multiplex ProcartaPlex immunoassay kit?

When combining a Multiplex kit with Simplex Bead Sets, a Basic kit is not required. All the non-target specific reagents which are included in the Basic kits are components of our Multiplex kits. Only when you are combining multiple Simplex kits (i.e., no Multiplex kit in the panel) will you need to also purchase a Basic kit.

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I accidentally stored the whole ProcartaPlex multiplex immunoassay kit at -20 degrees C. Can I use it anyway?

All the components included in the ProcartaPlex kit are viable except the bead mixture. Storage of the beads at temperatures below 0 degrees C will damage the beads and render them unusable.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What is the shelf-life of the ProcartaPlex multiplex immunoassay kits?

The ProcartaPlex kits are guaranteed until the expiration date written on the Certificate of Analysis. All kits are released with a minimum of 12 month shelf-life.

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For ProcartaPlex multiplex immunoassays, with serum samples and cell culture supernatant, I use 25 µL and 50 µL, respectively of recombinant standard to generate the top standard point. How is the standard curve same for both protocols?

When using serum/plasma samples with this assay, the protocol requires that 25 µL of sample be diluted with 25 µL of Assay Buffer. The top standard point is diluted in the same fashion, 25 µL of recombinant protein with 25 µL of Assay Buffer. As the dilution ratio between samples and standards is the same, we can use the same standard curve range. However, please note that the overall OD values might be a bit lower than in a cell culture supernatant assay.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What are the stopping points for the ProcartaPlex assay so I can incubate the plates overnight and continue with the run/analysis the next day?

There are two points at which the ProcartaPlex assay can be stopped and continued the next day:

- After the addition of standards and samples, the ProcartaPlex assay plate can be stored on a level surface overnight at 2-8 degrees C, covered, and protected from light. When you are ready to continue the assay the next day, bring the assay plate up to room temp on an orbital shaker (500-600 rpm), still protected from light, and continue with the assay protocol.
- After completing the assay, if the plate cannot be read on the Luminex instrument on the day of the assay, it can be covered and stored in the dark, overnight at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate the next day, bring the plate to room temperature on an orbital plate shaker (500-600 rpm) still protected from light. Perform a wash step using 100 µL of fresh, room temperature Working Wash Solution/Reading Buffer and perform the analysis. We do not recommend storing the ProcartaPlex assay plate longer than 1 day.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

For ProcartaPlex multiplex immunoassays, when preparing standards/samples, can I run them as single points?

For better accuracy, we recommend that you run samples/standards as duplicates at a minimum.

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For the ProcartaPlex multiplex immunoassays, where can I find the stock concentration of the recombinant standards?

This information can be found on the lot-specific Certificate of Analysis provided with the ProcartaPlex assay.

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Can I store reconstituted standard protein for later usage for ProcartaPlex multiplex immunoassays?

The recombinant standards included with the ProcartaPlex assay are one-time-use standards. Once reconstituted for use, we do not recommend storing and reusing this recombinant standard. With each new experiment, we recommend reconstituting a fresh unused vial of recombinant standard protein.

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For ProcartaPlex multiplex immunoassays, can I use a centrifuge for the washing steps in the assay?

No, we do not recommend centrifuging the magnetic beads for prolonged periods of time.

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For ProcartaPlex multiplex immunoassays, how are the standard recovery and bias values calculated?

When the MFI value of the standard value is used as an input, the concentration value that is generated can be used to evaluate the standard recovery. The standard recovery is calculated by taking the ratio of this calculated concentration value divided by the expected amount of standard and expressing that as a percentage. An acceptable range of recovery should be between 70-130%. This reflects a bias of 30%.

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What does "Limit of Quantitation" and “Limit of Detection/Sensitivity” mean for ProcartaPlex multiplex immunoassays?

The Limit of Quantitation ranges from the lower Limit of Quantitation (LLOQ) to the upper Llimit of Quantitation (LLOQ) The LLOQ and ULOQ are the lowest and highest analyte concentration that can be quantified with acceptable accuracy. The algorithms used to create the standard curves will impact the LOQ range. Depending upon the shape of the curve, a 5 parameter logarithmic curve fit (5PL) may yield better accuracy compared to a 4 parameter logarithmic curve fit (4PL).

In contrast, the LOD (limit of detection) or sensitivity is a calculated value determined in several independent assays. It is defined as the analyte concentration resulting in an absorbance significantly higher than the blank (mean of 6 independent assays plus 2 standard deviations).

There is no information about the lowest detectable concentration (lowest limit of quantification, LLOQ) from LOD/sensitivity.

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Does the labeling/marking of the plate have any influence on the measurement for ProcartaPlex multiplex immunoassays?

Waterproof markers that dry quickly will not affect the assay. However, markers that do not dry quickly might bleed into the wells during pipetting/washing which could influence the final readout.

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What type of analysis do you recommend for plotting the standard curve for ProcartaPlex multiplex immunoassays?

We recommend that you plot your data using a 4- or 5- parameter curve fit.

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For ProcartaPlex multiplex immunoassays, how many beads shoud I collect?

Please collect a minimum of 50 beads per bead region as stated in the user guide.

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Is it possible to measure ProcartaPlex Luminex beads on a flow cytometer?

No. To analyze ProcartaPlex (Luminex) plates, you will need access to a Luminex analyzer such as a Luminex 100/200, MAGPIX, or FLEXMAP 3D system.

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I have completed the ProcartaPlex multiplex assay but can't analyze the assay plate immediately. How long can I store the ProcartaPlex plate before analysis on a Luminex instrument without a significant loss of signal?

Ideally, we recommend that you analyze the samples immediately. If the plate cannot be read on the day of the assay, cover and store the plate in the dark, overnight at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate, bring the plate to room temperature on an orbital plate shaker (500-600 rpm), still protected from light. Perform a wash step using 100 µL of fresh, room temperature Working Wash Solution/Reading Buffer and perform the analysis. We do not recommend storing the ProcartaPlex assay plate longer than 1 day.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

During my ProcartaPlex assay data analysis, I am getting a warning message that there is high bead aggregation. What should I do?

Here are possible causes and solutions for this issue:

- Check the protocol settings (make sure you select the correct DD settings).
- Check the level of sheath fluid and empty the waste.
- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

During my ProcartaPlex assay data analysis, the beads fall below or to the lower left of their bead region on the bead map. Why is this?

Here are possible causes and solutions for this issue:

This usually indicates that the beads have been photobleached. This problem can also be caused by exposing the beads to organic solvents. Unfortunately, the assay will have to be repeated because the beads cannot be restored. The beads must be protected from light and organic solvents.
Alternatively, the instrument may be off in its measurements or you may have a calibration issue. Call the manufacturer for a service appointment.

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During my ProcartaPlex assay data analysis, beads do not appear in the region gated. What happened?

This indicates that an incorrect buffer was used for the final step. The Wash Solution provided in the kit must be used for washing the beads and the Reading Buffer should be used for resuspending the beads before loading them into the Luminex instrument. The osmolarity of the solution will impact the size of the bead, and any change in the bead size will alter detection by the instrument.

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The bead counts for all of my ProcartaPlex assay wells are erratic. What went wrong?

Here are some suggestions:

- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.
- Review the instrument settings and make sure they are appropriate for the assay being run (adjustment of needle height, make sure you select the correct bead gates and the correct DD settings).
- Shake the plate before acquisition on the instrument to resuspend the beads.
- Vortex the beads for 30 sec before adding them into the plate.
- Washing: Do not forget to keep the plate for about 2 mins on the Hand-Held Magnetic Plate Washer before emptying the plate.

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The bead count for my ProcartaPlex assay standard curve is correct and regular. The bead count for my samples is erratic and I get a warning message saying "The acquisition had at least one region that did not reach the maximum count". What should I do?

This pattern is indicative of a sample matrix effect. Here are some suggestions:

- Confirm that the sample has been clarified and is free of debris and free of lipids (5-10 min centrifugation recommended).
- Confirm that there is at least a 1:1 ratio of sample to assay diluent for serum, plasma samples. For cell lysates or tissue homogenates, confirm that the sample has been diluted appropriately in assay buffer to reduce the concentration of detergent in the lysis buffer to ⋜0.01%. For other sample types, further sample optimization may be required.

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The readout for my ProcartaPlex assay samples says that they are below the detectable limit. What are your suggestions?

Here are some suggestions:

- It is possible that the levels of your protein of interest fall below the detection limits of the assay. High Sensitivity Multiplex kits are available for most cytokines.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The readout for my ProcartaPlex assay samples says that they are above the detectable limit. What should I do?

Here are some suggestions:

- Sample optimization may be needed: Dilute the sample with an appropriate diluent and re-run.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How do I process cell lysates (extract cellular proteins) for the Luminex assay platform?

To process cell lysates (extract cellular proteins), follow the instructions provided here (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017835_PrepareCellLysates_Procartaplex_PI.pdf).

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How do I process saliva samples for the Luminex assay platform?

We have not specifically tested saliva samples in-house and therefore these instructions are only our recommendation. Saliva contains several proteolytic enzymes. It would be important to centrifuge samples and be sure not to pipet any cellular material or debris into the assay plate. We would suggest adding some anti-protease in the sample (for example, trasylol or aprotinine, 10 to 50 U/mL) to protect the protein from enzyme degradation. You may then centrifuge the samples at 1000 x g at 4 degrees C for 10 mins to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.

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How do I process oral mucosal transudate for Luminex assays?

We have not specifically tested oral mucosal transudate samples in-house and therefore these instructions are only our recommendation. Isolate the site around the tooth and insert a piece of periodontal filter paper into the gum pocket around the tooth for 30 seconds. Remove the filter paper and extract 4 times with 50 µL PBS for 5 min each at room temperature. The individual extractions can be combined and analyzed. Dilute 2-fold with Assay Diluent before applying to the assay.

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How do I process cervical secretion samples for use in a Luminex assay?

We have not specifically tested cervical secretion samples in-house and therefore these instructions are only our recommendation. Cervical sponges should be placed on ice immediately upon collection. Samples should be stored at -20 degrees C for up to one week and then stored at -80 degrees C until ready for assay. After thawing, sponges should be weighed and placed into Eppendorf tubes, using forceps cleaned with ethanol after each transfer. Add 200 µL of ice-cold extraction buffer (recipe below) to each tube and incubate overnight at 4 degrees C. The sponges and extraction buffer can then be transferred to microcentrifuge tubes with 0.2 µm cellulose acetate filters and centrifuged at 13,000 rpm for 10 min at 4 degrees C. The eluate can then be tested for cytokine expression.

Extraction Buffer
50 mM HEPES, pH 7.5
1 mM Na3VO4
150 mM NaCl
1 mM NaF
1 mM EDTA
0.1% Tween 20
25 mM EGTA
10% glycerol

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How do I process bronchoalveolar lavage (BAL) for use on the Luminex assay platform?

We have not specifically tested bronchoalveolar lavage (BAL) samples in-house and therefore these instructions are only our recommendation. The bronchoalveolar lavage (BAL) should be collected in a sterile syringe and kept on ice until you are ready to analyze it. Alternatively, BAL can be aliquoted and frozen in usable sample sizes (such that exposure to freeze-thaw is limited to one time). All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute 2-fold with Assay Diluent before applying to the plate.

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How do I process cerebrospinal fluid (CSF) for use on the Luminex assay platform?

Centrifuge samples at 1,400 rpm for 10 mins at 4 degrees C to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.

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How do I process synovial fluid for use on the Luminex assay platform?

We have not specifically tested synovial fluid samples in-house and therefore these instructions are only our recommendation. Collect synovial fluid into non-heparinized tubes and spin at 1,000 x g for 10 min within 30 min of sample collection. The acellular portion of synovial fluid should be stored at -80 degrees C before subsequent analysis. All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute samples 1:1 with Assay Diluent prior to addition to the assay. Reference: Raza K et al. (2005) Arthritis Research &Therapy 7(4): R784-R795.

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How do I process tissue homogenates for use on the Luminex assay platform?

Collect spleen, lung, brain, kidney, liver, or heart tissue and treat with or without LPS (100 µg, i.p., for 15 mins, 30 mins, 1 hr, 2 hrs, or 3 hrs). Weigh tissue in a 2 mL microcentrifuge tube. Add 500 µL of Cell Lysis Buffer (Cat. No. EPX-99999-000) per 100 mg of tissue. Add one 5-mm Stainless Steel Bead, then assemble tubes into TissueLyser according to the manufacturer’s recommendations. We recommend using 5-mm Stainless Steel Beads from Qiagen (Cat. No. 69989). Homogenize tissue at 25 Hz for 0.5-3 mins as indicated in the table below. Centrifuge the sample at 16,000 × g for 10 mins at 4 degrees C. Transfer the supernatant to a new microcentrifuge tube. Measure the total protein concentration. Dilute samples to 10 mg protein/mL with 1X PBS. To proceed with ProcartaPlex protocol, add 25 µL of Universal Assay Buffer (Cat. No. EPX-11111-000) to 25 µL of the diluted sample to each sample well.

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How do I process cell culture supernatants for use on the Luminex assay platform?

Cells should be in log-phase growth. Stimulate cells as desired in appropriate cell culture flasks. Using sterile technique, remove the desired volume of conditioned cell culture medium with a pipette and transfer the medium to clean polypropylene microcentrifuge tubes. Centrifuge the medium at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge to remove any cells or cellular debris. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes. These samples are ready for the assay. Alternatively, clarified medium samples can be aliquoted and stored at -80 degrees C for future analysis. Avoid multiple freeze-thaw cycles. Frozen samples should be allowed to thaw on ice just prior to running the assay. Thawed samples should be clarified by centrifuging at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge prior to analysis to prevent clogging of the Luminex probe and/or filter plate. Follow the assay procedure provided with the kit for appropriate dilutions.

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How do I process plasma samples for use on the Luminex assay platform?

Separate the cells from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete the sample of platelets. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2-8 degrees C while handling.

If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When you are ready to analyze them, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation (14,000 rpm for 10 min at 4 degrees C) in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.

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How do I process serum samples for use on the Luminex assay platform?

Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to clot for 20-30 mins at 20-25°C. Centrifuge at 1,000 x g for 10 mins at 20-25°C and collect the serum fraction. Alternatively, a serum separator tube can be used following the manufacturer's instructions. Use immediately or store aliquots at -80°C. Avoid multiple freeze-thaw cycles.

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Will ProcartaPlex multiplex assays give me the same results for each analyte as my current ELISA tests?

ProcartaPlex multiplex assays, which are based on Luminex xMAP technology, provide a versatile platform that gives users more flexibility and a greater array of options for analyte detection. Whether you are testing for single or multiple analytes, ProcartaPlex multiplex assays deliver accurate analytical performance using efficient, easy-to-follow protocols. Each of these assays has undergone the same development, validation, manufacturing, and quality control standardization we conduct for our ELISAs. Each lot of ProcartaPlex multiplex assays as well as ELISA assays is fully qualified with the appropriate sample type (i.e., species-specific serum, plasma, and cell culture supernatants), and each lot is evaluated based on the following performance characteristics:

Specificity-each analyte is screened to make sure there is no significant cross-reactivity with other analytes in the multiplex test
Sensitivity-each analyte is evaluated for both functional sensitivity (differentiation from background) and lower limit of detection (LLOD)
Precision/accuracy-multiplex assays have good intra-assay precision (<10% CV), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV); these values are comparable to or better than most ELISA tests
ProcartaPlex multiplex assays are regularly tested against the matching ELISAs. Therefore, you can switch easily from ProcartaPlex assays to ELISA and vice versa with reliable results. Most of our ProcartaPlex assays use the same antibody pairs as our traditional plate-based ELISAs, resulting in high correlation (R2 > 0.9) between the two assays.

Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.

How does the Luminex xMAP technology work for measuring more than one analyte in the same well?

Luminex xMAP technology is based on polystyrene or paramagnetic microspheres, or beads, that are internally dyed with red and infrared fluorophores of differing intensities. Each dyed bead is given a unique number, known as a “bead region”, allowing the differentiation of beads. For ProcartaPlex multiplex immunoassay kits, individual bead sets are then coated with a capture antibody qualified for one specific analyte. Multiple analyte-specific beads can then be combined in a single well of a 96-well assay to detect and quantify multiple targets simultaneously, using one of the Luminex instruments for analysis.

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What is the Luminex assay platform?

The Luminex assay is a bead-based immunoassay that uses beads of defined spectral properties conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration and test samples) into the wells of a microplate. The target protein binds to the capture antibodies over the course of a 2 hr incubation. After incubation on a shaker, the beads are washed by putting the 96-well plate on a flat magnet for 30 seconds, after which the fluid is discarded by flicking the wells or by using an automated plate washer. The magnet is removed, and the beads are resuspended in the detection antibody. Another incubation and wash are followed by the addition of streptavidin–R-phycoerythrin (SAPE). The beads are then washed and are ready to analyze. The Luminex technology is compatible with the following Luminex analyzers:

MAGPIX System-affordable, efficient, and compact
Luminex 200 System-versatile, efficient, and widely used in multiplexing
FLEXMAP 3D System-high throughput (up to 500 simultaneous assays) and automation compatible

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.