[Holsten]

Hi, guys.
I have a problem with viability of Jurkat's cells (T-cell derived cell line) in FACs analysis. Before staining I check viability by trypan blue and they look quite fine (only 3-5% of dead cells). But when I analyze them on cytometer up to 85-90% of cells are 7AAD positive. All procedures are performed on ice, pipetting is moderate, staining in total takes around 2 hours (1h-incubation with ligand, 20 min- antibodies, 15 min - 7AAD). I use standard FACS buffer: Mg free PBS, 5% horse serum, 2mM EDTA. Does anyone know what the problem can be? Thanks!

[CellSpecific.com]

First, what's the ligand? Second, try using PBS + 2% serum + 0.1% sodium azide as staining buffer. Hope this helps.

This post has been edited by Jay Dela Cruz PhD: 24 September 2009 - 07:13 PM

[Holsten]

Jay Dela Cruz PhD - Thank you for your reply.
Ligand is a half of the viral protein fused to Fc tag, it is known that it is not toxic, shouldn't do any harm to the cells. What is the advantage of using 0.1% sodium azide? As far as I know it can only prevent bacterial growth? Would it help to use 0.1-1% BSA instead of serum?

[CellSpecific.com]

Azide is often used to prevent "capping" during staining to maintain cell surface expression of markers. This is likely because azide inhibits mitochondrial respiration (http://www.pnas.org/...03/23/8646.full) and cuts intracellular ATP required in capping. You might find it interesting that some pathways to apoptosis are ATP-dependent. Hope things work for you.

This post has been edited by Jay Dela Cruz PhD: 25 September 2009 - 09:02 PM

[borriello.87]

Jay Dela Cruz PhD, on Sep 26 2009, 06:57 AM, said:

Azide is often used to prevent "capping" during staining to maintain cell surface expression of markers. This is likely because azide inhibits mitochondrial respiration (http://www.pnas.org/...03/23/8646.full) and cuts intracellular ATP required in capping. You might find it interesting that some pathways to apoptosis are ATP-dependent. Hope things work for you.

What do you mean by "capping during staining"?
I don't understand the meaning of these words....

Thanks!

[CellSpecific.com]

Capping is simply the redistribution of surface proteins due to antibody/protein complex formation that can result in shedding/loss of the complex. Here's a couple of background reading.

http://en.wikipedia....i/Cap_formation

http://www3.interscience.wiley.com/cgi-bin...434700/PDFSTART

[Holsten]

Thanks,guys, I used 0.1% sodium azide and it worked well, I had only 15% of dead cells in the end.
Another question is: what is better to use in FACS buffer: 0.1 -1% BSA, 5% horse serum or FSC?