Hello everyone, I use sorting to get T cells(CD4 and CD45RB double positive) and then culture in coating CD3(10ug/ml) U buttom 96 plate and treat the cell with soluble CD28(4ug/ml). After 3 days, I use cell titer blue to see T cell proliferation but there is no change in my experiment. Can anyone give me some advise?
The best way to check whether your T cells are proliferating or not is to look at the cells under a microscope. If your cells are proliferating, you would see huge clusters under the microscope (even at 2-3 days). This should tell you whether your aCD3/CD28 is activating the cells or not. If you see clusters but no measurable difference with cell titer blue (which by the way is a viability dye), try using a more sensitive T cell proliferation assay like thymidine uptake, CFSE dilution or Ki-67 staining. It could also be that you need to wait longer (5-6 days).
I will try wait for much long time and use CFSE to check the proliferation. Thank you very much!!!!
Dear Chiu Hsien Chan,
you should coat 96-well flat bottom plates and coat them with equal amounts of anti-CD3 (e.g. Orthoclone) and anti-CD28 antibody over night at 4° celsius or for 4 hrs at 37 °C. For the beginning, I recommend a concentration of 1µg/ml of each antibody. Please, be careful with anti-CD28 antibodies, only a few will have a stimulatory effect. After 5 days you should inspect your prolifarating T cell cultures. If you want to read out the proliferation I recommend a CFSE-labeling strategy, which is measured by flow cytometry.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating