Cell Cycle Analysis by
Propidium Iodide (PI) Staining
Adherent cells:
- trypsinized
- suspended
in medium + 10% FCS
- 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 (final concentration approx. 70%) - 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 –20°C).
Alternative
fixation with paraformaldehyde:
Pipet
the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r.t.
Staining:
- Pellet
the cells at 1500 rpm for 5 min
- Suspend
the pellet in 500 µl PI-solution
in PBS: 50 µg/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 37°C
- Add
3 ml of PBS, pellet the cells (1500 rpm, 5 min) and take off the supernatant
- Suspend
the pellet in 500 µl PBS for flow analysis
(you can also leave about 500 µl 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:
Approximate
settings (on FACSort):
FL1: 570 V log. (e.g. if you want to detect GFP)
FL2:
470 V linear
Example: