Search
Search
View additional product information for Calcein AM, Cell-permeant Green and Blue Dyes - FAQs (C1430, C3100MP, C481, C3099, C1429, C34852)
20 product FAQs found
一种可能性是染料之间的光谱渗漏。务必通过在绿光下成像红色细胞，在红光下成像绿色细胞的方式检查单色样品，对其他颜色使用最佳成像设置。如果您在对照中看到这些渗漏，那么就需要减少染料标记浓度以降低染料的亮度，或者选择那些光谱相距较远的染料。如果不是渗漏的问题，另一种可能性是细胞在染色后没有充分清洗，使得一些未结合的染料残留下来，这些染料进入后会标记其他细胞。延长清洗次数和时间应该会有帮助。
Calcein AM扩散到细胞中，“AM”部分由细胞酯酶裂解，随后染料分子的荧光信号可以在细胞质中被观测到，但其不结合到细胞组分。这意味着它们能够为“整个细胞”染色。这也意味着染料不能通过醛类固定剂进行交联，因此染料会在固定时丢失。此外，质膜的任何干扰（例如去垢剂或胰蛋白酶处理）都会导致染料从细胞中渗漏。
首先，确定您是在无血清条件下进行染色的。因为血清具有酯酶活性，会过早的裂解这些染料上的AM基团，进而阻止其进入细胞。染色之后即可将细胞移回含血清培养基中。除此之外，您可以尝试增加浓度和标记时间以获得更高的强度的信号。
钙黄绿素染料扩散到细胞中，“AM”部分由细胞酯酶切割，随后可以在细胞质中观察到而未结合任何胞内成分。这意味着它们标记了“整个细胞”。但是这也意味着它们可以通过正常细胞的外排机制被泵出。有时这一过程时间很短，尤其是显示出抗药性类型的细胞，除非外排被抑制（如使用丙磺舒抑制外排）。这也意味着染料不能被醛类固定剂所交联（不同于蛋白质结合CellTracker染料），因此染料会在固定时丢失。此外，质膜的任何干扰（例如去垢剂或胰蛋白酶处理）都会导致染料从细胞中渗漏。
用CellTrace试剂绘制出好的传代曲线的关键是在开始时均匀标记细胞，使它们在标记零点有紧密的变异系数（CV）。如果峰太宽，迭代相互重叠，一系列峰将成为一个驼峰。由于染色程度与细胞大小成正比，可以从统一的细胞群（而非淋巴细胞和粒细胞等混合的细胞群）开始标记，实现均匀的标记。细胞标记时间很短，所以您需要预先稀释染料，迅速混入你的细胞。请确保您的细胞未沉积在试管底部。实现这一点最简单的方法就是准备一份2X染色液（1×= 1-10μM），在一半体积（无血清或BSA）培养基中重悬细胞。将染料添加到细胞中并倒置几次以混合。染色过程中轻轻晃动细胞。染料孵育结束后（20分钟，37℃）即添加血清或BSA（至少1％），以清除任何残留的未反应染料。离心细胞，洗涤一遍，然后在完全培养基中重悬。经10-20分钟的脱酯化孵育后，细胞即可用于你们的实验。一定要确保有一个零点对照，因为您需要知道细胞第一次传代的时间。
可以通过流式细胞仪完成以下细胞健康和活力检测：
细胞凋亡检测：
细胞膜不对称性：膜联蛋白V是结构相关蛋白家族一员，其可以在Ca2+存在的情况下结合磷脂。膜联蛋白V可结合多种磷脂，但是对磷脂酰丝氨酸表现出高度的亲和性。磷脂酰丝氨酸主要存在于细胞膜的内部小叶上；然而，在细胞凋亡早期，观测到磷脂酰丝氨酸转移到外部小叶。这个转移使得在含有Ca2+孵育缓冲液存在的情况下，磷脂酰丝氨酸可与膜联蛋白V结合。凋亡中的细胞可以用膜联蛋白V染色，而正常细胞不会被染色。多种偶联不同荧光基团的膜联蛋白V可供选择。
线粒体健康：细胞凋亡早期的典型特征是线粒体紊乱，同时伴随膜和氧化还原电位改变。我们独家提供了大量可通过流式细胞术分析活细胞内的线粒体活性，同时可最大限度避免细胞功能损伤的荧光探针。
线粒体染色的MitoProbe系列染料（MitoProbe DiOC2(3) 检测试剂盒，货号 M34150；MitoProbe JC-1检测试剂盒，货号M34152；MitoProbe DiIC1(5) 检测试剂盒，货号M34151）为检测细胞凋亡过程出现的线粒体膜电位损失提供了快速、简单和可靠的流式细胞术检测手段。
半胱天冬酶活性： CellEvent Caspase-3/7 Green 流式细胞检测试剂盒（货号C10427）支持对凋亡细胞中活化的caspase-3和 caspase-7进行流式细胞术检测。该试剂盒包含新型荧光底物CellEvent Caspase-3/7 Green检测试剂和SYTOX AADvanced死细胞染色剂，可靶向识别活化的caspase-3和caspase-7的序列。
DNA片段化：细胞凋亡后期的特征是核形态改变，包括DNA片段化、染色质凝缩、核膜降解，核起泡以及DNA链断裂。凋亡过程中DNA片段出现DNA链断裂，可以通过TUNEL（末端脱氧核苷酸转移酶dUTP缺口末端标记）检测进行分析。APO-BrdU TUNEL检测（货号A23210）是一种双色检测方法，可通过成像或流式细胞术标记DNA断裂和细胞总DNA，检测细胞凋亡情况。
核染色质凝缩：细胞凋亡后期的特征是核形态改变，包括DNA片段化，染色质凝缩，核膜降解，核起泡以及DNA链断裂。凋亡的细胞表现出核染色质凝结增加。由于核染色质凝结，细胞透过性核酸染色剂发出较高的荧光，从而能够结合传统死细胞染色剂来区分凋亡细胞。
Vybrant细胞凋亡检测试剂盒#5，Hoechst 33342/Propidium Iodide（货号V13244）基于凋亡细胞染色质的压缩状态的荧光检测，为凋亡提供了一个快速和方便的检测手段。染色质凝结&膜渗透死细胞凋亡试剂盒包含Hoechst 33342、YO-PRO-1以及PI染料, 用于流式细胞仪（货号V23201）检测凋亡细胞中核染色质凝结和质膜通透性的变化。
细胞周期分析：
活细胞检测： Vybrant DyeCycle系列染料为活细胞周期分析提供了稳定的低毒性荧光染料，提供405 nm（货号V35003）、488 nm （货号V35004）、532 nm（货号V35005）或 633 nm（货号V10309和V10273）三种激发峰选择。染料毒性低，染色的细胞可储存和培养，或进行功能分析。
固定细胞检测：用FxCycle Violet染色剂（货号F10347）、SYTOX AADvanced 死细胞染色试剂盒（货号S10349）或FxCycle Far Red染色剂（货号F10348）分析细胞周期，为简单固定的细胞周期分析提供了多色选择。
细胞增殖：
染料稀释检测细胞增殖：染料稀释检测细胞增殖依赖细胞膜透过性荧光分子。染料进入到细胞后，共价结合蛋白质的氨基基团，导致染料长期保留在细胞中。通过随后的细胞分裂，每个子代细胞大约会分到亲代一半的荧光。采用流式细胞仪对细胞群的荧光强度进行分析，可以某个细胞或细胞群自标记之后的增殖情况，判定其传代次数。CellTrace荧光染色剂可在不影响细胞形态和生理功能的条件下，在体内或体外追踪传代情况。目前尚未发现该染色剂对细胞增殖活性或细胞生物学功能有影响。染色后，染料可在细胞内稳定保留若干天。可用于流式细胞仪的试剂盒包括CellTrace CFSE细胞增殖试剂盒（货号No. C34554）CellTrace Violet细胞增殖试剂盒（货号 C34557）和CellTrace Far Red细胞增殖试剂盒（货号 C34564）。
DNA合成检测：测定新合成的DNA是准确分析某个细胞或细胞群细胞增殖情况的方法。基于DNA合成的细胞增殖检测可根据掺入的修饰核苷，测定DNA合成速率。Click-iT Plus EdU细胞增殖检测利用了click化学试剂和修饰的核苷EdU，为BrdU染色提供了出色的替代方法，可用于检测和定量新合成的DNA数量。Pacific Blue（货号C10636）、Alexa Fluor 488（货号C10632和C10633）和Alexa Fluor 647（货号C10634和C10635）可用于Click-iT Plus EdU细胞增殖检测。
活力检测：
死细胞很容易与很多试剂非特异性结合，从而给出假阳性结果。因此，从流式细胞仪数据中排除死细胞，是有助于确保结果和分析准确性的关键步骤。
不能固定膜通透染色剂：SYTOX死细胞染色剂（货号S34857、S34860、S34861、S34859和S34862）不能穿过完整的细胞膜，与dsDNA结合后发出更强的荧光，从而成为我们最明亮的几种死细胞染色剂之一。非细胞通透性的经典DNA结合染料包括碘化丙啶（货号P21493）和7-AAD（货号A1310）。这两种染料已经被广泛用于流式细胞仪活性分析。CellTrace钙黄绿素AM染料可被动运输进入到贴壁和非贴壁细胞。这些细胞通透性酯酶底物可作为测定酶活性（激发荧光的必要条件）和细胞膜完整性的（在胞内保留荧光产物的必要条件）的活力探针。目前供应的蓝色（货号 C34853）、紫色（货号C34858）和绿色（货号C34852）荧光染料是活细胞短时染色的理想染料，且可用于多重流式细胞术试验。
可固定细胞活性染色剂：LIVE/DEAD可固定死细胞染色剂是可固定的细胞活性染料，有助于准确评估固定和/或通透后样品中细胞活性。LIVE/DEAD固定死细胞染色试剂盒基于荧光活性染料与细胞蛋白（胺基）的反应。这些染料不能透过活细胞膜，因此仅仅细胞表面蛋白可和染料反应，导致染色暗淡。活性染料可以透过死细胞损毁的细胞膜，将内部和外部的胺基染色，导致更加强烈的染色效果。LIVE/DEAD固定死细胞染色试剂盒可提供八单通道颜色，适用于三种包装规格的UV、405、488、532、561或633 nm激光，可满足您的试验需要。
•可测定来自单个细胞的数据。
•可从大量细胞中获得数据，产生细胞群的丰富统计学分析结果。
•由于可测定单个细胞，能够揭示种群的异质性。
•支持多重检测，可鉴定小型亚群。
•可以快速分析数以千计的细胞。
•非常适合血液样本和其他悬浮细胞。
•数据获取后，可以多次重复分析。
•流式细胞仪文件（FCS）可以归档。
可进行多种应用，包括免疫分型、细胞周期分析、凋亡检测（如膜联蛋白V染色检测实验）、CellEvent Caspase-3/7检测、TUNEL检测、细胞活性检测、增殖检测（如CellTrace 检测和Click-iT EdU检测）、MitoProbe检测法测定线粒体电势、利用计数微球进行细胞计数。
Yes. This is commonly done with calcein AM or FDA (fluorescein diacetate). These dyes will not fluoresce until cleaved by esterases. After modification by esterases and at very high concentrations, they will self-quench. Upon disruption of the plasma membrane, or cell death, the dye will be released into the extracellular medium, and become unquenched. Concentration and incubation time must be optimized to obtain adequate quenching.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Calcein, AM and FDA (fluorescein diaceate) are examples of some dyes used for this application. Since these dyes are not incorporated or covalently attached to any cellular components, they may have a short retention time as some cell types may actively efflux the dye out of the cells. The CellTracker and CellTrace dyes include either a mild thiol-reactive chloromethyl group or amine-reactive succinnimidyl ester group to allow for covalent binding to cellular components, providing for better retention. As with any reagent, one should empirically determine retention times for the cell type used.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Calcein AM, a green dye, is typically used as a general cytoplasmic stain, but not recommended with GFP-positive cells. For GFP-expressing cells there are other colors available: Calcein Blue AM, Calcein Violet AM, and Calcein Red-Orange AM. The retention time of these dyes in live cells is dependent upon the inherent properties of the cell.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Calcein, AM is a good choice for cell tracking and as a general cytoplasmic stain. However, it doesn't bind to anything and may be actively pumped out of the cells within a couple hours, which is likely what happened. The retention of Calcein within live cells is dependent upon the inherent properties of the cell type and culture conditions.
For long-term imaging, you may wish to consider a reactive cytoplasmic stains such as CFDA, SE or the CellTracker and CellTrace dyes.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
One possibility is that there is spectral bleedthrough between the dyes. Be sure to check the single-color samples by imaging the red cells in green and imaging the green cells in red, using the optimal imaging settings for the other color. If you see bleedthrough with these controls, then you will have to reduce the dye label concentration to reduce the brightness of the dyes, or choose dyes that are farther apart spectrally. If the issue isn’t bleedthrough, another possibility is that the cells were not adequately washed after staining, allowing some unincorporated dye to remain and label the other cells after they were introduced. Extending washes and wash times should help with this.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Calcein, AM diffuses into cells, the 'AM' moiety is cleaved by cellular esterases, and then the dye molecules are observed in the cytoplasm without binding to anything. This gives a 'whole cell' stain. It also means that the dyes are not crosslinked with aldehyde-based fixation and thus will be lost upon fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dye from the cell.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
First, make sure you aren’t staining in the presence of serum, since serum can have esterase activity that can prematurely cleave the AM group on these dyes, preventing entry into cells. After staining, it’s okay to return the cells to medium containing serum. After this, you can try increasing the concentration and label time to get a higher intensity.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Calcein dyes diffuse into cells, the 'AM' moiety is cleaved by cellular esterases and then are observed in the cytoplasm without binding to anything. This provides a 'whole cell' label. Calcein dyes may be pumped out by normal cellular efflux mechanisms, sometimes within a very short time, especially for cell types that may exhibit drug resistance, unless the efflux is inhibited (such as with probenecid). The dyes are not crosslinked with aldehyde-based fixation, unlike protein-binding CellTracker dyes, and thus will be lost upon fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dyes from the cell.
Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.
The key to good generational profiles with CellTrace reagents is starting with cells that are evenly labeled so that they have a tight coefficient of variance (CV) when run at time zero after labeling. If the peak is too broad, the generations will overlap each other and the series of peaks will become a hump. Even labeling can be achieved by starting with a uniform cell population (not a mixture of lymphocytes and granulocytes for example) as staining will be proportional to cell size. Cells are labeled rapidly, so you want to pre-dilute the dye and mix it into your cells rapidly. Be sure that the cells are not sitting in a clump in the bottom of your tube. The easiest way to do this is to make a 2x dye solution (1x = 1-10 µM) and resuspend your cells in a half volume of medium (no serum or BSA). Add the dye to the cells and invert a few times to mix. Gently agitate the cells during staining. Once the dye incubation is over (20 min, 37 degrees C), add serum or BSA (at least 1%) to scavenge any remaining unreacted dye. Spin down cells, wash 1x, and resuspend in complete medium. After a 10-20 min incubation to undergo de-esterification, cells are ready to be set up for whatever treatment you are planning. Be sure to keep a time zero control as you need to know where the first generation ran.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The following cell health and viability assays can be performed by flow cytometry :
-Apoptosis Assays:
Membrane Asymmetry: Annexin V is a member of a family of structurally related proteins that bind phospholipids in the presence of Ca2+. Annexin V binds several phospholipids, but shows highest affinity for phosphatidylserine.
Phosphatidylserine is normally found in the inner leaflet of the cell membrane; however, in the early stages of apoptosis, phosphatidylserine is observed to translocate to the outer leaflet. This translocation makes phosphatidylserine available for annexin V binding in the presence of Ca2+ containing incubation buffer. Cells undergoing apoptosis will stain with annexin V, while normal cells will not. annexin V is available conjugated with a wide range of fluorophores.
Mitochondrial Health: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We exclusively offer a number of fluorescent probes for analyzing mitochondrial activity in live cells by flow cytometry, with minimal disruption of cellular function.
The MitoProbe family of mitochondrial stains (MitoProbe DiOC2(3) Assay Kit, Cat. No. M34150, MitoProbe JC-1 Assay Kit, Cat. No. M34152, and MitoProbe DiIC1(5) Assay Kit, Cat. No. M34151) provides quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis.
Caspase Activity: The CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Cat. No. C10427) enables flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells. The kit includes the novel fluorogenic substrate CellEvent Caspase-3/7 Green Detection Reagent which targets the recognition sequence for activated caspase-3 and caspase-7, as well as SYTOX AADvanced Dead Cell Stain.
DNA Fragmentation: The later stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. DNA fragmentation that occurs during apoptosis produces DNA strand breaks, and can be analyzed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The APO-BrdU TUNEL assay (Cat. No. A23210) is a two-color assay for labeling DNA breaks and total cellular DNA to detect apoptotic cells by imaging or flow cytometry.
Nuclear Chromatin Condensation: The later stages of apoptosis are characterized by changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. Cells undergoing apoptosis display an increase in nuclear chromatin condensation. As the chromatin condenses, cell-permeable nucleic acid stains becomes hyperfluorescent, thus enabling the identification of apoptotic cells when combined with a traditional dead-cell stain. The Vybrant Apoptosis Assay Kit #5, Hoechst 33342/Propidium Iodide (Cat. No. V13244) provides a rapid and convenient assay for apoptosis based on fluorescence detection of the compacted state of the chromatin in apoptotic cells. The Chromatin Condensation & Membrane Permeability Dead Cell Apoptosis Kit with Hoechst 33342, YO-PRO-1, and PI dyes, for flow cytometry (Cat. No. V23201) detects apoptotic cells with changes in nuclear chromatin condensation and plasma membrane permeability.
-Cell Cycle Analysis:
Live cell assays: The Vybrant DyeCycle family of dyes offers robust fluorescent dyes for live-cell cycle analysis with limited cytotoxicity using 405 nm (Cat. No. V35003), 488 nm (Cat. No. V35004), 532 nm (Cat. No. V35005), or 633 nm (Cat. Nos. V10309 and V10273) excitation. The dyes have low cytotoxicity, allowing stained cells to be sorted and otherwise cultured or assessed with functional assays after staining.
Fixed cell assays: Analyzing cell cycle using FxCycle Violet Stain (Cat. No. F10347), SYTOX AADvanced Dead Cell Stain Kit (Cat. No. S10349) or FxCycle Far Red Stain (Cat. No. F10348) allows for multiple color options for simplified fixed cell cycle analysis.
-Cell Proliferation:
Dye dilution assays for cell proliferation: Dye dilution assays for cell proliferation rely on cell membrane–permeant fluorescent molecules. Upon entry into the cell, the dye will covalently bind to amine groups on proteins, resulting in long-term dye retention within the cell. Through subsequent cell divisions, each daughter cell receives approximately half the fluorescence of the parent. Analysis of the fluorescence intensities of cell populations by flow cytometry enables determination of the number of generations through which a cell or population has progressed since the label was applied. CellTrace fluorescent stains can be used without affecting morphology or physiology to trace generations in vivo or in vitro. There is no known effect on proliferative ability or biology of cells and they are well retained in cells for several days post-stain. Available kits for flow cytometry include CellTrace CFSE Cell Proliferation Kit (Cat. No. C34554), CellTrace Violet Cell Proliferation Kit (Cat. No. C34557), and CellTrace Far Red Cell Proliferation Kit (Cat. No. C34564).
DNA Synthesis Assays: Measuring the synthesis of new DNA is a precise way to assay cell proliferation in individual cells or in cell populations. DNA synthesis–based cell proliferation assays measure the rate of new DNA synthesis based on incorporation of modified nucleosides. The Click-iT Plus EdU cell proliferation assay utilizes the power of click chemistry and the modified nucleoside EdU to provide a superior alternative to BrdU staining for detecting and quantitating newly synthesized DNA. The Click-iT Plus EdU cell proliferation assay is available with Pacific Blue (Cat. No. C10636), Alexa Fluor 488 (Cat. Nos. C10632 and C10633), and Alexa Fluor 647 (Cat. Nos. C10634 and C10635).
-Viability Assays:
Dead cells often give false positive results, as they tend to bind non-specifically to many reagents. Therefore, removing dead cells from your flow cytometry data is a critical step to help ensure accurate results and analysis.
Non-fixable Membrane Permeability Stains: SYTOX Dead Cell Stains (Cat. Nos. S34857, S34860, S34861, S34859, and S34862) do not cross intact cell membranes, and they exhibit increased fluorescence upon dsDNA binding, making them some of our most brilliant dead cell stains. Cell-impermeant classic DNA-binding dyes include propidium iodide (Cat. No. P21493) and 7-AAD (Cat. No. A1310). Both of these dyes have been used extensively for viability assays in flow cytometry. CellTrace Calcein AM dyes can be passively loaded into adherent and nonadherent cells. These cell-permeant esterase substrates serve as viability probes that measure both enzymatic activity, which is required to activate their fluorescence, and cell membrane integrity, which is required for intracellular retention of their fluorescent products. Available with blue (Cat. No. C34853), violet (Cat. No. C34858), and green (Cat. No. C34852) fluorescence, these dyes are ideal for short-term staining of live cells and can be used in multiplexed flow cytometry experiments.
Fixable Viability Stains: The LIVE/DEAD Fixable Dead Cell Stains are fixable viability dyes that help to ensure accurate assessment of cell viability in samples after fixation and/or permeabilization. LIVE/DEAD Fixable Dead Cell Stain Kits are based on the reaction of a fluorescent reactive dye with cellular proteins (amines). These dyes cannot penetrate live-cell membranes, so only cell-surface proteins are available to react with the dye, resulting in dim staining. The reactive dye can permeate the damaged membranes of dead cells and stain both the interior and exterior amines, resulting in more intense staining. LIVE/DEAD Fixable Dead Cell Stain Kits are available in eight single-channel colors available for UV, 405, 488, 532, 561, or 633 nm lasers in three packaging sizes to match your experiment.
-Measures data from single cells.
-Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
-Because single cells are measured, it will reveal heterogeneity within a population.
-With the ability to multiplex, small sub-populations can be identified.
-Thousands of cells can be analyzed rapidly.
-It is ideally suited for blood samples and other cells in suspension.
-Data can be re-analyzed multiple times after acquisition.
-Flow cytometry files (FCS) can be archived.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace assay and Click-iT EdU assay, measurements of mitochondrial potential with MitoProbe assays, and cell counting using counting beads.
Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.