I have an idea about the overview of the test, however, I need step-by-step details to follow in carrying out the test.
Testing CD4 and CD8 T cell responses in splenocytes using flow cytometry involves a series of steps to prepare, stain, and analyze the cells. Below is a detailed protocol for this procedure:
**Materials:**
1. Splenocytes (isolated from mouse spleen)
2. Appropriate cell culture medium (e.g., RPMI-1640)
3. Fetal bovine serum (FBS)
4. Penicillin-streptomycin solution
5. Phosphate-buffered saline (PBS)
6. Red blood cell lysis buffer (if working with mouse splenocytes)
7. CD4 and CD8 fluorescent antibodies (fluorochrome-conjugated)
8. Cell surface marker antibodies (e.g., CD3, CD45)
9. Fixation and permeabilization buffer (e.g., BD Cytofix/Cytoperm)
10. Intracellular cytokine staining antibodies (e.g., IFN-γ, IL-2, TNF-α)
11. Appropriate isotype control antibodies
12. Flow cytometry tubes or plates
13. Flow cytometer equipped with appropriate lasers and filters
14. Software for flow cytometry data analysis
**Procedure:**
1. **Isolation and Preparation of Splenocytes:**
a. Sacrifice the experimental animals or obtain spleen tissue from human donors following ethical guidelines.
b. Isolate splenocytes using standard procedures. If working with mouse splenocytes, use red blood cell lysis buffer to remove erythrocytes.
c. Wash the isolated splenocytes with PBS and resuspend in culture medium (e.g., RPMI-1640) supplemented with FBS (10%) and penicillin-streptomycin (1%).
2. **Stimulation and Activation:**
a. Plate the splenocytes at a density of 1-2 million cells per well in a 96-well plate.
b. Add a T cell activation cocktail that includes an appropriate mitogen or antigen (e.g., PMA and ionomycin) and a protein transport inhibitor (e.g., GolgiStop) to prevent cytokine secretion.
c. Incubate the cells for 4-6 hours at 37°C in a CO2 incubator to allow for T cell activation and cytokine production.
3. **Surface Staining:**
a. Wash the activated splenocytes with PBS to remove the activation cocktail.
b. Incubate the cells with fluorescently labeled antibodies against surface markers, including CD4, CD8, CD3, and CD45, according to the manufacturer's instructions. Use appropriate isotype control antibodies as controls.
c. Incubate for 30 minutes in the dark at 4°C.
d. Wash the cells with PBS to remove unbound antibodies.
4. **Fixation and Permeabilization:**
a. Fix and permeabilize the stained cells using a fixation and permeabilization buffer according to the manufacturer's protocol. This step is necessary for intracellular staining of cytokines.
5. **Intracellular Staining for Cytokines:**
a. Incubate the fixed and permeabilized cells with fluorescently labeled antibodies against intracellular cytokines (e.g., IFN-γ, IL-2, TNF-α) in the dark at 4°C.
b. Incubate for 30 minutes, following the manufacturer's recommendations.
6. **Flow Cytometry Analysis:**
a. Analyze the stained splenocytes on a flow cytometer equipped with appropriate lasers and filters.
b. Gate on lymphocytes based on forward and side scatter characteristics.
c. Analyze CD4 and CD8 T cell responses by examining the expression of surface markers and intracellular cytokines.
d. Collect data for a sufficient number of events (e.g., 10,000-50,000 events) for statistical analysis.
7. **Data Analysis:**
a. Use flow cytometry analysis software to quantitate CD4 and CD8 T cell responses, including the percentage of positive cells and mean fluorescence intensity (MFI) of cytokine expression.
b. Compare experimental samples to appropriate controls (e.g., unstimulated cells, isotype controls) to determine specific T cell responses.
8. **Interpretation:**
a. Interpret the flow cytometry data to assess CD4 and CD8 T cell responses based on the expression of surface markers and intracellular cytokines.
9. **Reporting:**
a. Compile and report the results, including the percentages and MFI values for CD4 and CD8 T cell responses, as well as any relevant statistical analyses.
Remember to follow biosafety and ethical guidelines throughout the procedure, and adjust the protocol as needed based on the specific requirements of your experiment and the flow cytometry equipment available in your laboratory.
Hi Mark Ige
I briefly wanted to reach out concerning your question.
For a complete guide to splenocyte restimulation including experimental protocols for restimulation and flow cytometry including optimized staining for both intra- and extracellular proteins please see:
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
To assess CD4/ CD8 lymphocyte activation ex vivo, please use:
Article Ubiquitin Specific Protease 21 Is Dispensable for Normal Dev...
And for a comprehensive 48-plex cytokine/ chemokine analysis for mouse samples (erum, plasma, cell culture supernatant or cell pellets) please consider:
https://olink.com/products-services/target48/t48-mouse-cytokine/
I hope that helps.
All the best & kind regards,
Michael
Thank you Doctor Lukmnan.
This is well composed, it will surely benefits alots. Thank you very much
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