The most accurate way is to add 7AAD or propidium iodide and eliminate dead cell from analysis. If you need to fix samples, I once used FVS 620 from bd with excellent results, and they have several different colors to choose.

Hi Diego and George:

Thanks for joining this discussion. I proposed this question because i re-looked my previous 10 color FACS data, quite often there is a population looks suspicious, even though i excluded dead cells by fixable live/dead cells marker from Invitrogen. Meanwhile, i found this is very general phenomenon when i googled FACS plot, quite often we can see something looks worrying in the published data, some of them from very prestigious journals.

George, i am not sure whether the dead cell number maybe the reason, but i should try it. Thanks.

Best regards

Shiqiu

Hello

Yes , the FACE by MACS® Dead Cell Removal Kit it's a good choice .

https://www.miltenyibiotec.com/LV-en/products/macs-sample-preparation/removal-reagents/dead-cell-removal-kit.html

Good Luck

I once found a population that was 7AAD negative but I suspected they were dead cells. I asked people with more experience in flow citometry and they told me they probably were early apoptotic cells. Those cells are negative to 7AAD but they are determine to die and sometimes they interact differently with antibodies. Those cells have a relative high SS but very low FS. I attached a copy of how I removed those cells form analysis.

Hope it helps

Hi Diego:

Thanks for sharing. I totally agree with you.

7-AAD mostly stain necrotic cells by passing through the big pore on the cellular membrane, and intercalate with DNA, just like PI. These necrotic cells are late stage dead cells, cells are shrink and more particles inside, so they showed low FSC and high SSC; For apoptotic cells (early dead cells), their membrane is still intact, and 7-AAD can not get in, but their nucleus start to condensate and shrink, cell size get smaller and contents get denser. In most cell death assay, Annexin V+7-AAD can split all dying cells into 3 different populations.

For this reason, if we use 7-AAD as the only dead cell marker, there is quite a chance to include some dying cells into live cell population. This may be one of the source of dying cell contamination. As you showed above, careful gating on scatter plot can avoid this.

Best regards

Shiqiu