如果您不仅使用了Panel Builder配色工具 ,还期望与流式科学家共同设计复杂流式Panel,赛默飞在全球遴选出一位位优秀的流式技术支持科学家,加速您的流式实验。我们深刻理解完成复杂流式配色十分花费时间、且劳心费神。虽然赛默飞不能代替做完实验,但流式科学家可以与您一起设计流式方案,充分考虑荧光溢漏,实现更加清晰的细胞分群。在使用这个Panel时,我们始终提供技术支持,优化流式Panel,得到最佳结果。点击 赛默飞科学家流式配色,联系我们,开始配色。

开始使用Panel Builder流式配色

使用Panel Builder,可以轻松选出最佳荧光染料搭配。流式小白和大神都在用的Panel Builder流式配色工具可以与您一起轻松五步完成流式配色,加速流式实验进程。

教学视频:如何轻松上手Panel Builder流式配色工具

观看小片,了解简单5步如何轻松上手Panel Builder流式配色工具,设计您的流式抗体方案。

教学视频:如何设计光谱流式配色

观看小片,开始您的光谱流式配色旅程

五步轻松玩转Panel Builder流式配色

刚刚开始流式实验的小白?多年流式摸索经验的大神?Panel Builder都可以帮助您轻松五步选择流式仪器、抗原、荧光、抗体及最后总结,得到满意结果。

选择流式仪器



选择抗原





完成所有选择后,单击 Next Step(下一步)。

选择流式荧光染料

流式荧光染料选择指南主要基于每个荧光染料的光谱信息。在这里,当您选择了每个抗原的荧光染料后,可以直接在页面顶部看到荧光染料的光谱信息。

在选择荧光染料之前,每个抗体都显示了可用的检测通道。每当选定每个抗体后,页面顶部会直接显示其荧光光谱。在这里,可以看到每行是列出的抗原、每列是显示的通道。

在Step 2中选择的蛋白表达丰度会在这里推荐相应荧光染料,有小黑角标做提醒。




光谱流式实验配色

复杂指数

最后总结


想再查看荧光溢漏?单击激光下的 View Spectra 即可查看光谱。

还不满意现在方案?您可以单击 Edit panel 返回编辑, 或者可以单击页面底部返回到前面步骤中。在使用过程中,可以点击进度栏返回前面步骤中。 

当您检查完每一步、还很满意结果,流式配色也将要完成了。确定保存了您的流式配色结果,并且为其命名。这里可以选择以Excel或PDF下载流式配色结果数据,方便查看或打印。

最后,轻松添加您的流式Panel到购物车中,方便下单。在Step 5中直接显示您所选的抗体价格;单击 “Add all to cart”,这里所选的抗体都将添加到购物车中。

Invitrogen Flow Cytometry Panel Builder FAQs

  1. Can I go back a step without losing the work I did?
    • Yes, the previous work is saved.
  2. My marker isn’t showing up under step 2 of antigen, but I see it in the catalogue.How can I remedy this?
    • Select your target species first, and then type your antigen.
  3. How do I share my panel with my lab manager?
    • Export the spreadsheet found on step 5.
  4. What do the colorful dots indicate in the “Fluorochromes” step?
    • The dots indicate the number of fluorochromes that Thermo Fisher Scientific has available in each channel on the cytometer you have chosen.
  5. My cytometer does not use a standard configuration.How can I make changes to one in the dropdown?
    • Select a cytometer from the dropdown menu and then select the “Edit cytometer settings” link to modify the configuration as needed.
  6. I can’t find my cytometer in the dropdown.Can I build one?
    • Yes, you can define a new cytometer using the “Enter your cytometer manually” link below the cytometer dropdown menu.
  7. I do not order products through thermofisher.com.What other endpoints are available to me?
    • There are both PDF (printable) and CSV (spreadsheet) exports available in the summary step for you to take away for reference and ordering purposes.
  8. I plan to use antibodies that are not from Thermo Fisher Scientific.How can this Panel Builder be useful to me?
    • Step 2 allows for antibodies you already purchase or from outside Thermo Fisher Scientific.If you are undecided about which format you want for something, you can add the antigen name in the “Antibodies you need” section and then choose a placeholder fluorochrome in the following step.Both will help you keep track of channels that are reserved for reagents that you do not plan to buy from Thermo Fisher Scientific.
       

流式荧光染料选择指南

流式荧光发射光时有不同的亮度(图 2)。在Panel Builder上选择荧光染料时,赛默飞建议:

  • 基于特定流式细胞仪,测试染色指数。流式厂家在他们的仪器上使用同一克隆、不同荧光基团的抗体测试出染色指数。也可以用流式微球得到染色指数,比如图 2 显示了在Attune NxT流式细胞仪上常见荧光染料的染色指数。每个流式细胞仪都有不同的激光、滤光片和检测通道,这都会影响到荧光染料偶联的标志物检测信号。
  • 根据蛋白表达水平,搭配荧光染料的不同亮度。 仅仅使用非常明亮的流式荧光会导致荧光溢漏,并减弱标志物信号。
  • 在每种抗体克隆及其荧光染料上,检测其平均荧光强度来比较亮度。这些可以在产品数据表中找到。

想要了解更多染色指数信息?想要查看BD LSRII 流式细胞仪上的染色指数?请点击: 流式细胞术多色方案设计:基础知识

蛋白表达丰度(抗原密度)

了解您的蛋白表达丰度,可以更好地确定相应流式荧光染料。非常明亮的流式荧光染料适用于低表达丰度的靶标, 较暗的荧光染料更适合高表达丰度的靶标, 赛默飞建议:

  • 基因表达水平会因细胞来源、刺激条件和固定方案而变化。
  • 很暗的荧光染料可以检测高表达蛋白,而很亮的荧光染料方便检测低丰度蛋白。
  • 使用流式补偿微球可以帮助确定抗原表达强度。各大基因遗传数据库也可以确定RNA表达水平。
     

最佳流式配色的快速技巧

&赛默飞流式科学家 Natalie Oxford 分享了她的体会,尤其是在发表级流式配色上。
 

想要查看更多配色技巧?请点击:流式细胞术多色方案设计:基础知识

赛默飞流式荧光染料列表,包含其优点和类型。

了解荧光染料的激发光谱和发射光谱,请使用Invitrogen SpectraViewer

类别系列优点Invitrogen流式荧光染料
有机染料:小分子、且稳定天然染料
  • FITC的性价比高
FITC
Pacific dyes
  • 部分亮度是最暗的
Pacific Blue
Pacific Orange
Alexa Fluor dyes
  • 光稳定性强,在可见光光谱上都有
  • 用于流式分析和成像细胞分析
  • 以其激发波长命名
Alexa Fluor 405
Alexa Fluor 488
Alexa Fluor 532
Alexa Fluor 561
Alexa Fluor 647
Alexa Fluor 660
Alexa Fluor 700
eFluor 有机染料
  • 用于流式细胞分析
  • 以其发射波长命名
eFluor 450
eFluor 506
eFluor 660
蛋白类大分子染料天然染料
  • 性价比高
  • 部分是最亮的染料
APC
PE
PerCP
串联染料
  • 串联的两个供体分子占着多个检测通道,可以用于构建更大的流式Panel
APC-Cyanine5
APC-Cyanine7
PE-Cyanine5 (TRI-COLOR)
PE-Cyanine5.5
PE-Cyanine7
PE–Texas Red
PerCP-Cyanine5.5
PE–Alexa Fluor 610
PE–Alexa Fluor 700
APC–Alexa Fluor 750
PE–eFluor 610
PerCP–eFluor 710
APC–eFluor 780
聚合物染料Super Bright流式荧光染料
  • 由405 nm紫激光激发
  • 避免荧光溢漏到其他通道
  • 使用两种或更多的聚合物染料时,添加Super Bright Complete Staining Buffer 货号No. SB-4401-42) 来降低背景噪音影响。
Super Bright 436
Super Bright 600
Super Bright 645
Super Bright 702
Super Bright 780
Brilliant Ultra Violet 流式荧光染料
  • 由紫外激光激发
  • 用于流式细胞分析
Brilliant Ultraviolet 737
Brilliant Ultraviolet 805
DNA结构染料NovaFluor 流式荧光染料
  • 用于流式细胞分析
  • 以其激光激发和发射波长命名
  • 充分避免激光交叉激发
NovaFluor Blue 510
NovaFluor Blue 530
NovaFluor Blue 555
NovaFluor Blue 585
NovaFluor Blue 610-30S
NovaFluor Blue 610-70S
NovaFluor Blue 660-40S
NovaFluor Blue 660-120S
NovaFluor Yellow 610
NovaFluor Yellow 660
NovaFluor Yellow 690
NovaFluor Yellow 700
NovaFluor Yellow 730
NovaFluor Red 660
NovaFluor Red 685
NovaFluor Red 700
NovaFluor Red 710

Tip 1: Minimize spill-over by using the correct and separate channel

Any time you have markers that you know will be co-expressed on your cells of interest, make sure to space them out into separate channels.If you will need to use any adjacent channels, that's where you would put any markers that are mutually exclusive so that they'll still be easy to distinguish.

Tip 2: Intracellular targets need special buffer for fixation and permeabilization for staining

You'll also want to keep in mind the buffer that you're using to fix and permeabilize your cells, as we have several options.When you're looking at cytoplasmic targets, what the buffer is appropriate may not be the same as when you're looking at nuclear targets, because you want to make sure that you still have access to your antigens without over-fixing your epitopes.

Tip 3: Viability dyes are required to find live cells

A third tip I wanted to share with you is to always include a viability dye in your staining panel.This will help eliminate any false positives that are caused by dead cells or debris, because those can be sticky.You have a lot of options for choosing a viability dye, so you don't need to design your panel around them.You can build out the rest of your panel and optimize your core markers, and then fit in a viability dye in an empty channel.

Tip 4: Save your bright fluorochromes for dim targets

As you're building out your basic panel and you want to incorporate some more antigens, make sure you're keeping the density of your antigen expression in mind.So if you have antigens with low or unknown expression, those would be ones that you want to assign to your brightest dyes, such as PE or APC.

Tip 5: Try to combine negative markers in one channel (dump channel) to save space on your panel

A helpful trick when you want to exclude a lot of cell types at once without having to suck up multiple channels for that would be to use a dump channel.This is where you're placing all the antibodies that identify your cells that are not of interest into the same channel with the same fluorochrome, and then those can be easily gated out and all of the cells negative for the dump channel would be those that you use for your analysis going forward.

技术

学习

不得转售。Super Bright 聚合物染料是在Becton, Dickinson and Company的许可下销售。
Brilliant Violet和PE-CF染料为Becton, Dickinson and Company专有。
Cy™ 是Amersham Biosciences Corp的商标。Cy 染料为Amersham Biosciences Corp和Carnegie Mellon大学专有,并且依据 Amersham Biosciences Corp 的许可下进行生产和销售,仅用于研究和体外诊断用途。
Brilliant Violet™ 是 Becton, Dickinson and Company 或其附属公司的商标或注册商标,并在其许可下使用。由 Sirigen™ 提供支持。

仅供科研使用,不可用于诊断目的。