Cell Cycle Analysis with DAPI by stariya

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									                         Cell Cycle Analysis with DAPI
                            Current Protocols in Cytometry


Reagents:

80% EtOH (store at -20oC)

DAPI/TX-100 Solution:
0.1% Triton X-100 (stock 1%; 1 ml for 10 mls final)
1 ug/ml DAPI (Invitrogen D1306)(stock 1 mg/ml in water, store at -20oC; 10 ul for
10 mls final)
in PBS (9 mls for 10 mls final)
Make fresh.

Protocol:

Keep cells on ice prior to fixation.
Aliquot 2x106 cells into a FACS tube.
Wash 2X with cold PBS.
Decant the last wash and resuspend pellet prior to the addition of EtOH.
Resuspend cells in 1 ml 80% ice-cold EtOH while vortexing.
Incubate on ice a minimum of 30 minutes. (Cells can be stored at 4oC in EtOH for
several months).
Wash cells 2X in cold PBS.
Resuspend cells in 1.0ml freshly-made DAPI/TX-100 solution and incubate 30
minutes at room temp.
Filter samples to remove aggregates immediately prior to analysis.

Excitation/Emission:
Ex. 405nm
Em. 450/50 BP




                  Harvard Systems Biology Flow Facility 2009 JKM

								
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