Hi everybody!

I'm a student looking fore some suggestions on how to stimulate in a TCR-Dependent manner my cells to assess early phosphorylations (SLP-76, LAT and SHP) in TCR signalling cascade through Phospho-flow.

I tried some ''classics'' but I can't figure out what I'm doing wrong. Here some protocolos I've already used:

- Anti-CD3 + Anti-CD28 on ice for 15 minutes followed by Purified Goat Anti-Mouse on ice for 15 minutes and then 5 minutes at 37 Celsius. Then Washing Buffer and centrifuge and then Fixation;

- Same thing, but only Anti-CD3 or Anti-CD3+Anti-CD28 (without Purified Goat Anti-Mouse phase)

- Same thing, but with Anti-CD3/Anti-CD28 Beads

Please help me I'm going mad.

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