Frustrated with FACS (ONLY DEAD CELLS) - (Aug/02/2007 )
I have been trying FACSing for nearly 1 months without a single positive result. I am working with Hep2 cells which is an adherent immortalised epithelial cell line. I wanted to look at the apoptosis with and without a drug. The protocol i followed is as follows
60mm confluent cells split on the previous day. On the day of FACS cells incubated in the presence and drug for 4h followed by removing the media completely, trypsinizing with 0.1% trypsin for 15 sec after which media with FBS added to neutralize the trypsin. Cells removed by pipetting, pelleted at 1200rpm for 5 min at 4 degrees. media removed and washed with 1X PBS twice. Cells resuspended in 500microlitres 1X PBS, to which 10 microlitres (1mg/ml) propidium iodide added. kept on ice for 2 h depending on the travelling time to the institute where FACS is kept. Plus or minus 1hr depending if the person incharge is available or someone else is analyzing the sample. after analysis i am informed that DNA has fragmented and only dead cells in both control and treated samples.
can someone please help me out
!) If there is some problem with my protocol ? any step needs modification
2) Has anyone faced this problem before?
I think you may have incubate the cells with propidium iodide for too long.
Try to reduce the incubation time to 30min.
Hope this may help.
I have not worked with that cell line, but I have done flow on several cell lines and I can tell you that I always see the cells die if I dont' add FBS (2%) into the PBS used both for washing and resuspension of the cells. you may wish to try that as well?
As suggested: reduce time for staining and add FBS, but I was wondering if you could not do PI staining, wash your cells and then fix them (e.g. in PBS with PFA)? (I have never done PI-staining myself so this might be the stupiedest suggestion you could get).
Thanks aimikins, minnie thats a good suggestion i will try that out. I was wondering if i could skip adding media to trypsinize the cells if i am adding PBS with FBS?
Vairus i have read about fixing with ethanol and then staining for long term storage of samples for FACS, but i dont know if i should try that. If i try that i will let u know if it worked.
Is it necessary to incubate with PI for 2h?
PI can stain well in 15 mins. If U suspect PI doing the damage, you can instead carry PI along to the place where U do FCM and stain it right there before you submit for FCM.
Fixing the cells is a good idea and can keep cells stable for weeks, right?
if i am looking at Cell Cycle and apoptosis; i usually fix 1x10^6 cells (previously washed in PBS) in ice cold 70% ethanol (usually overnight, but 2 hours is good), pellet the cells, wash twice in 1x PBS, and then resuspend my pellet in 500 - 1000 ul of propidium iodide. This generally produces very good results using our FACS Calibur.
Thankyou all for the suggestions. This time i added 2% FBS to my PBS and carried out the washing steps and it worked! also i reduced my PI incubation time (carried out the staining in the institution where the FACS is kept) my one month jinx finally broken.
thank u guys
Good to hear that it worked.
I was wondering why you don't use FACS buffer to suspend cells.