PE Annexin V Apoptosis Detection Kit with 7-AAD

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells werepretreated with OVA-257 peptide SIINFEKL for the indicated time.	- cells were co-activated by anti-CD3 and anti-CD28 Abs for the indicated time

Upstream tips
- Cells werepretreated with OVA-257 peptide SIINFEKL for the indicated time.
Protocol tips
- cells were co-activated by anti-CD3 and anti-CD28 Abs for the indicated time

Publication protocol

Cell proliferation and apoptosis rates were determined by flow cytometric analysis. For the analysis of proliferation, miR-491-overexpressing T cells and mock cells were isolated by density gradient centrifugation and labelled with carboxyfluorescein diacetate succinimidyl diester (CFSE) (5 μM, eBioscience) for 10 minutes at room temperature, and the reaction was stopped with one volume of foetal calf serum. Then, the cells were washed three times with PBS. These cells were continually cultured for another 48 hours under standard conditions, and the proliferation rate was assessed. For the apoptosis experiment, miR-491-overexpressing T cells and mock cells were co-activated by anti-CD3 and anti-CD28 Abs for the indicated time. OT-I T cells after transfection were isolated and restimulated with the OVA-257 peptide SIINFEKL for the indicated time. Then, cells were double-stained by annexin V/7AAD (BioLegend), and the rate of apoptosis was assessed. The results were analysed by FlowJo software version 6.2.

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