Hello all,
I could really use some advice with T cell intracellular cytokine staining. I've been trying to stimulate CD4+ T cells isolated from mouse spleens and stain intracellularly for cytokines, including IL-2, IFN-gamma, and IL-17A. However, I see very poor cytokine staining in my stimulated T cells, even IL-2 staining. I expected that most of my stimulated T cells would be positive for IL-2 intracellular staining, but only about 3 percent of my T cell population are positive for IL-2-PE when I run my samples on our flow cytometer. I see clear activation of my T cells not treated with Brefeldin A based on increased percentages of T cells positive for CD69 post stimulation, so I doubt that stimulation is the problem. If anyone has tips or tricks on optimizing T cell intracellular staining to share, then that would be most helpful!
Below is my general protocol:
1) Isolate CD4+ T cells from mouse spleen by mashing spleens and using StemCell EasySep CD4+ T cell Isolation Kit on the spleen lysate (I usually get 3-6 million T cells depending on the age and genotype of the mice)
2) Plate CD4+ T cells in 24-well TC-treated plate coated with anti-mouse CD3 antibody (5 ug/mL); soluble anti-mouse CD28 antibody is added (5 ug/mL); usually 400,000 T cells in 1 mL of R10 solution
3) Incubate T cells for 30 minutes at 37 degrees Celsius
4) Add 1 microliter of Brefeldin A (1,000x stock solution) to wells; I've also tried 0.5 microliters
5) Incubate T cells for 6-12 hrs at 37 degrees Celsius; wash after incubation with PBS/BSA
6) Stain cells for cell membrane markers (CD4, CD25, CD69) for 30 minutes
7) Incubate cells in BD Cytofix/Perm for 20 minutes; wash with BD Perm/Wash
8) Stain cells with intracellular cytokines (e.g. IL-2-PE) diluted in BD Perm/Wash for 30 minutes (1:50 antibody dilution)
9) Wash cells with BD Perm/Wash
10) Resuspend cells in 1xPBS 1%BSA
11) Ran cells on BD Accuri Flow Cytometer, collecting 50,000-100,000 events
12) Analyzed flow data and performed compensation using FlowJo software
Hi Carlos,
I would try to incubate the cells with only CD3/CD28 in R10 for a bit longer (try 3 days) and then follow up with 1x Brefeldin in fresh medium for 5 hours.
You may also try to add Monensin (Similar to Brefeldin), but I´d first go with a longer CD3/CD28 incubation.
Lastly: If that doesn´t work, try CD3/CD28 beads, for example from Thermo Fisher. Use a ration of ~1 bead/cell. These beads are exprensive, but much more effective than CD3 coated plates.
Best regards
Johannes
Hi Carlos,
My name is Sneha and I am a Product Manager at STEMCELL Technologies.
As Johannes mentioned, I would increase the incubation time. I would recommend stimulating with CD3/28 for 24 hours and re-stimulating the cells with PMA/Ion and Bref A for the final 4 hours. The PMA/Ion re-stimulation will help make more IL-2. Also of note, within this time frame, it might be challenging to see IL-17 without the addition of polarizing cytokines.
Please find a methods article linked here referencing times and concentrations of CD3/28, Brefeldin A and PMA/Ion : https://www.frontiersin.org/articles/10.3389/fimmu.2022.759188/full
Please don't hesitate to reach out to us at [email protected] if you have any further questions. Good luck :)
Thanks,
Sneha
I used to stimulate the cells for 24 hours at 37C with 5% CO2.
Add 1 ul 1x Brefeldin in fresh medium incubate for 5 hours at 37C with 5% CO2. The cells were washed three times with staining buffer and then continued with an intracellular staining procedure. Cells surface staining was performed and incubated at 4 ºC for 30 minutes in the dark. The cells were then washed three times, and 250 µl of cytofix/cytoperm reagent was added followed by incubation at 4 ºC for 15 minutes. The cells were washed with cytoperm buffer three times prior staining with intracellular conjugated antibodies stain.
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