Cell Cycle Analysis (DAPI) with Fluorescent Protein Detection
Current Protocols in Cytometry 7.16
This protocol is compatible with cells expressing nuclear and/or cytoplasmic fluorescent
proteins.
Reagents:
1X PBS (Phosphate-Buffered Saline)
70% Ethanol, pre-chilled at -20 C.
Paraformaldehyde (PFA) fixation solution
1X PBS
1% PFA
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:
1. Trypsinize adherent cells to detach, and wash once in 5-10 ml 1X PBS to remove
residual serum and trypsin. Cell number per sample should be 1-2x106 cells.
2. Resuspend each cell pellet in 1ml PFA fixation solution; incubate on ice for 1
hour.
3. Spin cells at 300g, 5 min, 4 C. Remove PFA, wash cells once in 5-10 ml 1X PBS.
4. Resuspend each cell pellet in 0.5 ml 1X PBS. Vortex tube gently and add 4.5ml
ice cold 70% ethanol dropwise over 30 sec to 1 min. Incubate cells at 4 C
overnight (minimum 2 hours).
NOTE: At this step cells can be stored at -20 C for up to several weeks.
5. Spin cells at 300g, 5min, 4 C. Remove supernatant. Wash cells twice in 5-10 ml
1X PBS.
6. Pellet cells, 300g, 5 min, 4 C.
7. Remove supernatant; resuspend cells in 0.5 ml DAPI/TX100 staining solution (or
1X PBS solution for –DAPI negative controls).
8. Incubate 30 min at RT.
9. Filter and analyze.
Harvard Systems Biology Flow Facility 2009 JKM