Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
- - - - -

staining at 37 degrees or RT - OK or really bad ?

staining protocol temperature

  • Please log in to reply
3 replies to this topic

#1 alwaysbreizh



  • Members
  • Pip
  • 4 posts

Posted 06 October 2011 - 09:21 PM


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 ?


#2 Rsm


    Post Dog

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 385 posts

Posted 07 October 2011 - 01:19 AM

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.
I got soul, but I'm not a soldier

#3 RynDggn



  • Active Members
  • Pip
  • 14 posts

Posted 28 November 2011 - 08:02 PM

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.

#4 science noob

science noob


  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 284 posts

Posted 29 November 2011 - 01:12 PM

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".

Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.