Intracellular Protein Staining with Cell Cycle Analysis (DAPI)
Adapted from Cytometry 71A:905-914, 2007
Reagents:
Paraformaldehyde (32% Solution, EM Grade, MeOH-free) (EMS Cat # 15714)
Dilute to final concentration in 1X PBS.
FACS Buffer
PBS with 1% FCS/FBS
80% EtOH (store at -20oC) Can substitute 100% MeOH (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:
Harvest cells.
Keep cells on ice prior to fixation.
Aliquot 2x106 cells into a tube.
Wash 2X with cold FACS Buffer. Decant Supernatant.
PF Fixation
 Resuspend cells in 1ml of 1% paraformaldehyde solution and incubate for 15
minutes on ice. (Longer incubations compromise the quality of CV’s in DNA
histograms.)
 Wash 2X with cold FACS Buffer.
EtOH Fixation
 Decant the last wash and resuspend pellet in remaining residual volume
prior to the addition of EtOH.
 Resuspend cells in 1 ml of 80% ice-cold EtOH, added slowly while vortexing
the sample.
 Incubate on ice a minimum of 30 minutes. (Cells can be stored at -20oC in
EtOH for 2-24 hours).
Harvard Systems Biology Flow Facility 2009 JKM
Antibody Staining
 Wash 2X with cold FACS Buffer. Decant Supernatant completely. Pellet will
become translucent following EtOH-fixation and will be difficult to visualize.
 Incubate cells in 100ul final volume of directly conjugated antibody(ies),
diluted to recommended concentration in FACS Buffer, for 30-60 minutes on
ice.
 Wash cells 2X in cold FACS Buffer. Decant Supernatant.
DAPI Staining
 Resuspend cells in 0.5ml freshly-made DAPI/TX-100 solution and incubate
30 minutes at room temp.
 Immediately prior to analysis, filter samples to remove aggregates.
For a good review of Intracellular Protein Staining methodologies (including nuclear
permeabilization and alternative fixation protocols) see Cytometry 55A:61-70,
2003.
Harvard Systems Biology Flow Facility 2009 JKM