BioProtocols--cell10

Cell Cycle Analysis by PI Staining/FACS Protocol 1.

Fixation

1) Collect 1 X 10⁶ cells after proper treatment.

2) Pellet cells by spinning at 2,000 rpm, 4C for 5 minutes.

3) Resuspend cell pellet in 1 ml of cold FCM buffer (5mM EDTA in 1xPBS) .

4) Fix cells by adding 1 ml of -20C absolute ethanol.

5) Store cells at -20C in this fixation buffer until ready for analysis. (No more than 2 weeks)

Staining

6) Centrifuge (as above) fixed cells and resuspend pellet in 1 ml of FCM buffer.

7) Add 2 ul mg/ml DNase-free, RNaseA and incubate at 37C for 20 minutes.

8) Add 1 ul of 5 mg/ml propidium iodide (light sensitive) and incubate at room temperature for 15 minutes.

9) Pellet the cells by spinning at 2,000 rpm, 4C for 5 minutes.

10) Wash with FCM buffer once more, discard the buffer and resuspend the stained cells with

500 FCM buffer.

11) Place samples in 12 X 75 Falcon tubes and read the data (% of G0 G1 G2 MS) on a FACS analysis system.

Cell Cycle Analysis by PI Staining/FACS Protocol 2.

Adherent cells:

trypsinized

suspended in medium enriched with10% FBS

centrifuged (1000 rpm, 5 min)

Pellet suspended in PBS (1 ml)

Suspension cells:

Centrifuged (1000 rpm, 5 min)

Pellet suspended in PBS (1 ml)

Fixation with EtOH:

Pipet cell suspension into 2.5 ml absolute EtOH - or vortex the suspension at half speed while adding the EtOH) - to prevent clustering of cells during the fixation. Incubate on ice for 15 min (or over night at -20C).

Alternative fixation with paraformaldehyde:

Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at room temperature.

Staining:

Pellet the cells at 1500 rpm for 5 min

Suspend the pellet in 500 ul PI-solution
in PBS: 50 ug/ml PI from 50x stock solution (2.5 mg/ml)
0.1 mg/ml RNase A
0.05% Tritin X-100
Incubate for 40 min at 37C

Add 3 ml of PBS, pellet the cells (1500 rpm, 5 min) and take off the supernatant

Suspend the pellet in 500 ul PBS for flow analysis
(you can also leave about 500 ul of the diluted staining solution on the pellet and suspend the cells in this solution > less loss of cells when you take off the sup.) -the rest of the staining solution does not interfere with the flow analysis.

Flow analysis:

Reference settings (on FACSort):
FL1: 570 V log. (if you want to detect GFP)
FL2: 470-480 V linear

Other Cell Cycle Analysis Protocols