Xxxxxxx V staining for apoptosis Sample Clauses

Xxxxxxx V staining for apoptosis. Induction of apoptosis was investigated using annexin V conjugated to FITC. 3-6x104 cells were seeded in 12 well plates in complete medium + 10% FCS. Cells were treated for the appropriate time (e.g. 24-72h) and harvested with trypsin as discussed. Cells were transferred to 5ml polystyrene tubes (BD biosciences) and centrifuged at 2000xg for 5 mins. Media was poured away and cells were washed once in 500μl Annexin V binding buffer (BD Biosciences) and again centrifuged. Excess buffer was poured off, and cells were resuspended in remaining buffer (≈100μl). 2μl of Annexin V-FITC were added and 4μl of PI solution (50mg/ml stock) and cells were incubated for 20 mins on ice in the dark before adding a further 100ul of PBS and acquiring using a FACS Canto II.
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