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Annexin V and 7-AAD flow cytometry analysis

JC Jennifer S. Chambers
TB Tim Brend
TR Terence H. Rabbitts
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At 48 hrs post-doxycycline induction, cells were harvested and pelleted at 200 × g for 5 mins, room temperature. 5 × 104 cells were washed and resuspended in 100 μl in Annexin V buffer (10 mM HEPES pH7.4, 140 mM NaCl, 2.5 mM CaCl2) with 2 μl Annexin V-FITC (BD Pharmingen) and stained on ice for 30 mins. Cells were washed and resuspended as before, with 5 μl 7-AAD (50 μg/ml, BD Pharmingen) and incubated on ice for 15–20 mins diluted with annexin V buffer immediately before collecting fluorescence measurements on an Attune flow cytometer (Invitrogen). Data was analyzed using FlowJo software (TreeStar).

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