3 replies to this topic

[alwaysbreizh]

Hi,

Several antibodies require staining at 37 degrees (chemokine receptors)  or eventually RT (tetramers) and I noticed doing that, that most of the time every other markers in my panels look brighter and 'cleaner'.

I've always been told that phenotypic stains have to be performed at 4 degrees one reason for that being to limit cell death. From my experience, staining at 37 degrees or RT for no longer than 30 minutes, does not affect cell viability or only marginally (looking at pretty much every cell populations in mouse spleens, LN, BM, liver). So I was wondering if there was other rational reasons underlying why stains have to performed at +4 ?

Thanks.

[Rsm]

My experience is that it significantly decreases background. Anyway, why would keeping the cells on ice prevent cell death? I would assume that keeping them at body temperature (37C) prevents cell death better than at RT or 4C. Usually, you keep the cells on ice to prevent changes in signaling pathways due to disruption from the niche.

[RynDggn]

The reason for keeping the cells on ice is to reduce internalization of the antibody.  If cells are metabolically active and going through their normal surface antigen turnover, you could internalize antibodies which could lead to increases in background.  One way to go about your staining procedure would be to stain the chemokine receptors 1st, wash, then do subsequent surface staining on ice or 4C and then wash again.

[science noob]

I've always wondered about the difference between primary antibody incubation conditions.  I was told that 37 is optimal since that's the normal temperature natural antibodies would be in our bodies.  So, has it been proven that +4 would change signalling pathways?

Also, if you've fixed your cells, all cells are somewhat "dead".