[민유홍] P.I. staining for cell cycle assay (for FACS)

from Yu-Hong Min

< P.I. staining for cell cycle assay>

ž  P.I. staining Sol.

                                                                       stock       1ml     2ml       5ml                                

                    50ug/ml P.I.                      10mg/ml      5ul      10          25

                    0.1% Sodium Citrate           10%        10        20          50

                     0.3% NP-40                          10%         30       60         150

                       50ug/ml RNase A           10mg/ml       5        10           25                                

                        in 1X PBS                                            950     1,900     4,750

ž  Staining

1.     Culture the cells (70-80% confluent in 35mm dish for just 1 sample).

2.     Adherent cells: collect floating cells in the medium (which consist of detached mitotic, apoptotic, and dead cells) and trypsinized cells (after trypsinization, must add medium containing serum to inactivate trypsin) into a 15ml conical tube.

Suspension cells: transfer cells in medium into 15ml conical tube.

3.     Centrifuge 3min at 1500rpm, room temperature in a swinging rotor (for apoptosis analysis, centrifuge 10min at 300 x g).

4.     Remove supernant.

5.     Add 0.3ml of PBS, then resuspend cells thoroughly by pipetting (use yellow tip).

6.     Add 0.7ml of cold 100% ethanol, then vortex.

7.     Incubate on ice for ≥15 min or over night at –20°C (cells in 70% ethanol can be stored at –70°C for several months).

8.     Centrifuge as in step 3.

9.     Remove supernant.

10.  Suspend pellet in 1-2ml PBS by vortexing, wait 1min, then centrifuge as in step 3. Decant supernatant.

11.  add 400ul(or 500ul) of P.I. staining sol. then resuspend cells thoroughly by pipetting (use yellow tip).

12.  incubation for 20-30min at 4C.

13. NO Wash!!!!!!!

14.  Directly FACS reading.