Cell Storage Conditions for Cell Cycle Analysis - PI staining with GFP containing construct (Feb/02/2008 )
I am in the process of setting up a cell cycle analysis on K562 cells transfected with my gene and GFP (under a separate promoter) using PI. I'm using the information found in the references below in order to do this experiment without losing the GFP during the permeabilization process. I will do the experiment over a time course of 5 days (removing a sample of cells each day) and would like to do the analysis at the end of the 5th day. My question is the proper way to store the cells. I am going to use 1% PFA to fix the cells followed by 70% EtOH permeabilization. I've read that you can store the cells but they do not provide many details.
These are the options that I have come with up. Should I:
1) After fixing do the PI staining the day (or day after) that I collect the sample and then store samples at 4C in EtOH
2) After fixing do the PI staining the day (or day after) that I collect the sample and then store samples at 4C in PBS
3) Fix and permeabilize the cells and store at -20C in EtOH and then do staining the day for all of the samples the day of analysis
4) Fix and stain the cells and store PI staining solution at 4C until analysis
5) Fix and stain the cells and do the analysis on that sample every day (would be tedious and more expensive to do this way so I would rather store and run all at once)
Any other suggestions or conditions that anyone can provide!
Thanks so much!!
Analysis of DNA Content and Green Fluorescent Protein Expression - Curr Protocols in Cytometry, Unit 7.16.2
Analysis with flow cytometry of green fluorescent protein expression in leukemic cells. - Cytometry. 1999 Aug 1;36(4):333-9. Chu et al.
Here's what I do.... the key is to be gentle so as not to fragment the dehydrated cells after fixation
Day of Harvest
1) Trypsinize cells-->collect cells in stale media-->spin-->(wash w/ice cold PBS-->spin)x2-->
2) resuspend cells in cold PBS adjust cell concentration to 1x10^6/ml, hold on ice. Transfer 1ml to round bottom FACS tubes
3) Slowly add an equal volume of ice cold absolute EtOH to cell suspension to fix/permeabilize
4)***KEY STEP*** gently mix cell suspension by slowly blowing bubbles through a pasteur pipette attached to pipet-aid
5) Cap the tubes and wrap with parafilm to prevent evaporation they can be stored several weeks at -20C (will not freeze becasue of EtOH)
Day of FACS
1) bring cells to room temp, then centrifuge at ~ 300-500xg for 5 min
2) slowly add room temp PBS to cells and incubate at 37'C for 15 minutes to rehydrate-->centrifuge at ~ 300-500xg for 5 min
3) Resuspend cell in 1 ml PBS with RNAse A (200ug/ml final) and incubate for another 15-30 mintues-->centrifuge at ~ 300-500xg for 5 min
4) Resuspend pellet in Citrate-Saline with PI (D'oh...I'm forgetting the [PI] right now), and hold cell on ice, in dark
5) perform FACS within 3 hours
Thanks! Do you have any hints for working with paraformaldehyde? I have to fix with PFA first so that I can cross link the GFP inside the cells to decrease GFP leakage and then permeabilize with EtOH. Is using 100% EtOH better than using 70% or it doesn't make a difference? Or is 100% EtOH better for storage?