Dear Silvana,
Most annexin kits recommend the use of the following buffer:-
HEPES buffer:
10mmol/l HEPES
To 100 mls diluted HEPES Add:- NaCl 876mg (150 mmol/l)
KCl 37.3mg (5
mmol/l)
CaCl2 26.5mg (1.8
mmol/l)
NaOH to pH 7.4 MgCl2 9.5mg (1 mmol/l)
Method
1) Harvest cells, wash in PBS, count.
2) Add 0.5x106 cells per 12mm test tube, wash in 1 ml Annexin Buffer and
discard supernatant.
3) Wash again in 1ml Annexin Buffer, discard supernatant.
4) Mix to break cell pellet and add 5ul Annexin V/FITC. Incubate 4oC 15
minutes.
5) Wash in Annexin Buffer, discard supernatant.
6) Add 10ul PI solution (10ug/ml in PBS) and 190ul Annexin Buffer. Mix
gently.
7) Analyse on the flow cytometer
Flow cytometry
Use dual parameter analysis for PI staining (FL2 or FL3, red optics
>600nm) Log. scale on the x-axis, versus FITC Annexin staining (Log.
scale FL1 530nm bandpass filter), y-axis.
Good luck,
John
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