Annexin V-FITC Detection

(1) incubate cells with treatment in 15 ml tubes (Tube 1 (DCs only - control), Tube 2 (heat killed DCs  (cells boiled at 70C for 20 min) + control), Tube 3 DCs + EGCG50, Tube 4 DCs + ECCG100, Tube 5 DCs + Lp, Tube 6 DCs+Lp+EGCG50, Tube 7 DCs + Lp + EGCG100)

(2) transfer cell cultures next day to glass tubes and place on ice. For + control transfer some from cell only (preferably 1 ml, ottherwise split the cell only into 0.5 and 0.5) into a 2 ml tube. place the tube in hot plate for 2' at 87C. Then transfer the contents of + control to glass tube (blue cap refer # 35204).

(3) Add about 5 ml cold PBS to cell cultures and spin down (cntrifuge 5 ')

(4) repeat wash with 5 ml cold PBS

(5) resuspend cells in 1X binding buffer at a concentration of 1x10⁶ cells/ml. So if you have 1x10⁶ cells/ml simply add 1 ml

(6) for each treatment have 2 glass tubes (for unstained and stained PI+annexin). Transfer 100 ul of the solution from (5) to each of these tubes (the untreated sample you may want to have 4 tubes for "unstained, stained annexin, stained PI and stained PI+annexin")

(7) add 5 ul of Annexin V-FITX and 5 ul of PI to the stained tubes

(8) gently vortex the cells and incubate for 15 min at RT (25C) in the dark (hood above centrifuge).

(9) Add 400 ul of 1x binding buffer to each tube.

(10) analyze by flow cytometry within one hour. (change blank for sample FACS tube. Mix tubes on rack before putting in holder!)

Ex.     tube #        1        3        5        7        9  ---- unstained

         tube #        2        4        6        8        10 ----stained

                    DC only +Lp  +EGC10 50    100