Hello,
I am using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain from Thermofisher (https://www.thermofisher.com/order/catalog/product/L34965), to distinguish dead cells. I use it on freshly isolated human PBMCs and on CD4+ T cells (with ~90% viability).
When I try to compensate, whenever I gate on my lymphocyte population, I dont have enough positive events (dead cells) for the dye.
At first, I have been told to gate on my lymphocyte population and include events at the at the lowest SSC and FSC values (which I thought they correspond to debris and dead cells), but then I have been told that is not a good way to do it because those event correspond to autofluorescence.
I read (I can't remember where) that dead cells are usually located at low FSC and high SSC, is that true?
There is also the possiblity to try to add to my compensation tube dying cells (after trying to freeze them maybe). Is this possible? Is it the only way?
Thank you,
Martin
Hi Martin:
It is not a good idea to squeeze live cells with debris, which are not cells any more, just small piece or shrink nuclei containing particles. Besides their high autofluroscence induced technique problems, they also bias your biological observations.
To induce cell death, there are 3 main ways:
1) Physical methods: such as 60 degree heat treated cells, then mix with live cells;
2) Chemical methods: many compound can induce apoptosis, such as etoposide.
3) Biological methods: over-activated cells will die, such as PMA+Ionomycin.
By the way, if you are not working on multi-colour FACS (such as 10 or more) and your FACS machine equipped with 3 or 4 laser, you can establish your Ab panel properly, making this live/dead cell marker has little spillover to others, in this case, you can tick off this color from your compensation panel.
Best regards
Shiqiu
Hi Martin,
I think the way I usually choose when my samples have very high viability, and I need samples for live/dead compensation is to leave some of the cells at 50-60 degree Celsius for ~10-15 mins, then mix them with live cells.
Hope this helps, and best of luck with your work.
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