Practice English Speaking&Listening with: How to Characterize Immune Responses With Flow Cytometry

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- [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

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

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.

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.

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