
Chinese: 
如何使用流式细胞术
表征免疫应答
包括实验设计、抗体选择
及对亚群设门
如果您面临挑战
需要使用流式细胞术
设计抗体检测组合
来分析细胞内信号转导
而您不确定该怎么办
没有关系
我们可以帮您做到
我们来看看一个实验的例子
向您展示如何使用
多重靶标的流式细胞术
构建一个抗体检测组合
来评估免疫细胞激活
您还可以访问网站
cst-science.com/youcanflow
下载配套应用说明
多重靶标测定能够增强
流式细胞术分析效果
表型表面标记物
可以精确地找到哪些细胞群
对处理或实验干扰有反应
同时通过胞内读数
还能检测与这种反应有关的
信号转导通路
在我们的示例中
我们的目标是

English: 
- [Instructor] If you are
faced with the challenge
of designing antibody panels to analyze
intracellular signaling
with flow cytometry
and you're not sure how to
find your way, that's okay.
You can do it, we can help.
We will walk through an example experiment
to illustrate how you can
build an antibody panel
for multiplex flow cytometry
to assess immune cell activation.
You can also download the
companion application note,
at cst-science.com/youcanflow.
Multiplexing adds power to
your flow cytometry analysis.
Phenotypic surface markers pinpoint
which cell populations
exhibit responses to treatment
or experimental pertubation
while intracellular readouts
probe the signaling pathways
involved in that response.
In our example experiment, our
goal is to assess activation

Chinese: 
在异质PBMC样本中
评估T淋巴细胞的激活
您可选择适当的表面标记物
来开始构建检测组合
这样您能够在分析中
对细胞群和亚群设门
在我们的示例中
用CD3作为总的T细胞标记物
用CD4作为辅助性T细胞标记物
添加一个或多个胞内靶标
作为信号转导通路激活
蛋白表达
或其他生物功能的读数
我们用磷酸化特异性抗体
来检测SLP76的磷酸化
作为T细胞激活的读数
最后
考虑在你的抗体检测组合中
加入一种鉴定活/死细胞的染料
例如Ghost Dye
从而能够从分析中排除死细胞
下面是一些
确保你的流式细胞术结果
可靠且可重复的最佳做法
在搭配你的抗体检测组合时
确保选择

English: 
of T cells lymphocytes within
a heterogeneous PBMC sample.
Start building your panel by selecting
appropriate surface markers so
that you can gate populations
and sub-populations in your analysis.
In our example, we used
CD3 as a pan-T cell marker
and CD4 as a helper T cell marker.
Add one or more intracellular
targets as readouts
for signaling pathway activation,
protein expression or
other biological functions.
We used a phospho-specific antibody
to detect phosphorylation of SLP-76
as a readout of T cell activation.
Finally, consider incorporating
a live/dead viability stain,
such as a Ghost Dye, in your
panel to allow exclusion
of dead cells from analysis.
Here are some best practices
to ensure your flow cytometry
results are reliable and reproducible.
When building your panel, make sure
to select antibodies
or antibody conjugates

English: 
that have been validated
in flow cytometry.
Performance in other
applications does not tell you
how that antibody will perform in flow.
It's also important to
check that all antibodies
in your panel are
compatible with the fixation
and permeabilization conditions
used in your protocol.
Testing and optimizing of protocol steps
may be required if this
information isn't available.
Finally, when selecting fluorophores,
avoid spectral overlap
to the extent possible,
or incorporate fluorescence compensation
into your analysis when necessary.
To prepare our live PBMCs for
flow cytometry and analysis,
we first incubated cells
with Ghost Dye and washed.
Then, we activated the
T cells by treating them
with antibodies to induce
cross-linking
of CD3 and CD28 for 15 minutes
prior to fixation, permeabilization,
and labeling with antibodies for analysis.

Chinese: 
已经经过流式细胞术验证的
抗体或抗体接合物
其他应用中的性能
不能代表
该抗体在流式细胞术中的表现
检查你抗体检测组合中的
所有抗体是否适合您实验中
使用的固定和通透处理条件
如果没有此类信息
可能需要测试并优化实验步骤
最后，选择荧光素时
尽可能避免光谱重叠
或在必要时向您的分析中
加入荧光补偿
为流式细胞术分析
准备活PDMC细胞
我们先用Ghost Dye
孵育细胞并清洗
随后
我们用交联CD3/CD28处理
激活T细胞
15分钟后进行固定
通透处理并用抗体标记
为了在T细胞中分析SLP76活动

Chinese: 
我们应用序贯设门方案
在门1中使用前向/内向散射
来选择淋巴细胞
在门2中用Ghost Dye
选择活/死细胞
随后在门3中
针对CD3表达进行选择
在门4中针对CD4表达进行选择
用这种方法设门
能够让您快速聚焦于相关细胞群
并获得重要的数据
在我们的实验中
T细胞激活
在CD3/CD4阳性T细胞中
清晰可辨认
并且在设门细胞群中
激活后
磷酸化SLP76表达呈阳性的
细胞比例高于一般PBMC
如需更详细的指南
来设计您的流式细胞术检测组合
请访问cst-science.com/youcanflow
下载应用说明

English: 
To analyze SLP-76 activity in T cells,
we employed a sequential gating scheme.
Forward- and side-scatter were used
in Gate One to select lymphocytes.
The live/dead selection with
Ghost Dye was used for Gate Two
followed by selecting for
CD3 expression in Gate Three
and CD4 expression in Gate Four.
Gating in this way enables
you to quickly zero in
on relevant cell populations
and get to the data that matters.
In our experiment, T cell activation
is clearly distinguishable
in CD3/CD4 positive T cells,
and the percentage of cells positive
for phospho-SLP76 expression
after activation is higher
in the gated cell population
than in the bulk PBMCs.
For a more detailed guide to designing
your flow cytometry panel,
visit cst-science.com/youcanflow
and download the application note.
