I am going to measure T cell activation in different conditions using Anti-Phosphotyrosine by Flow cytometry.
I am going to inmovilized Ab-CD3 on wells, put my T cells on it and then stain them for FACS.
I would like to know how to do the dynamic via CD3-TC. Do I need a time course of 1-5-10 min?
Thanks in advance
Hi, the time points you mention are fine, but maybe technically difficult to analyze in your settings (eg with plate bound antibodies). Likely not all the cells will encounter the stimulatory antibody especially for the 1min time point.
You may rather try to stimulate with soluble anti-CD3. Alternatively , you may first incubate your cells on ice with anti-CD3, then add a crosslinking antibody at 37°C to aggregate the receptors. The latter approach should yield stronger phosphorylation but also faster extinction of signaling.
If possible, I suggest adding an intermediate time point between 1 and 5.
Good luck!
Hi, the time points you mention are fine, but maybe technically difficult to analyze in your settings (eg with plate bound antibodies). Likely not all the cells will encounter the stimulatory antibody especially for the 1min time point.
You may rather try to stimulate with soluble anti-CD3. Alternatively , you may first incubate your cells on ice with anti-CD3, then add a crosslinking antibody at 37°C to aggregate the receptors. The latter approach should yield stronger phosphorylation but also faster extinction of signaling.
If possible, I suggest adding an intermediate time point between 1 and 5.
Good luck!
Hi Alfonso,
Vincenzo is right about the technical difficulties of using plate-bound anti-CD3e. One alternative is to plate the cells at 4ºC and then perform a centrifugation to force the system a little. Doing FACS of these cells at different time points (after fixing and a gentle permeabilization) should give you a higher sensitivity than WB (the clone PY20 is a very good one for mouse T cells).
Cheers,
Dear Alfonso,
I agree with Vincenzo and Pablo on the technical challenges. With plate-bound Abs, not all cells will come into contact with anti-CD3. Centrifugation works well in this setting, however we also use Ab-coated Dynabeads Pan mouse IgG (you need to optimize the concentration of anti-CD3 on these beads as you don't need a lot). We use 4beads/cell and make a brief centrifugation (low speed) of cells+beads followed by incubation for 3min then separate beads from cells using a magnet within 2extra minutes (total time of contact C+B in our case is 5min), then start staining at 4C.
Good luck,
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