Cell Cycle Analysis (PI) 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
NOTE: PFA must be heated to >65 C to dissolve. Helps to add 1-2 drops 10M
NaOH during mixing.
PI staining solution (Store at 4 C protected from light for up to one month)
1X PBS
100 ug/ml RNAse A
40 ug/ml propidium iodide
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 1-2x10
6
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 PI staining solution (or 1X PBS solution for –PI negative controls).
8.
Incubate 30 min at 37 C.
9.
Filter and analyze.
Harvard Systems Biology Flow Facility 2009 JKM