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!
Photo
- - - - -

Thawing PBMCs

cell culture Flow ELISpot

  • Please log in to reply
3 replies to this topic

#1 teddytariku

teddytariku

    member

  • Active Members
  • Pip
  • 9 posts
1
Neutral

Posted 10 September 2011 - 12:41 PM

Hi Everyone

I am having huge problem in recovering cells from HIV/TB patients which is stored in -80 freezer for the past 1 and half years. I am planning to perform ELISpot, Flow analysis and monocyte infection with Mycobacterium tuberculosis. Unfortunately, my cell yield is very low compared to what I have frozen initially. When consulting, people told me that I can use DNase, but the stuff that we have is a molecular biology grade and I have to use higher concentration. Moreover, I haven't seen much difference when using this enzyme with the cells for 1hr at 37oC in 5% CO2. I am requesting those of you who have similar experience to share how I would solve or reduce PBMC clump formation.

Thank you very much for your help in advance!

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,828 posts
414
Excellent

Posted 14 September 2011 - 09:58 PM

When storing cell lines you should store them in liquid nitrogen. Most cells will die quite quickly at -80, this is especially true for primary cells such as your PBMCs!

There is not much you can do about the death, you'll just have to work around it.

#3 teddytariku

teddytariku

    member

  • Active Members
  • Pip
  • 9 posts
1
Neutral

Posted 19 September 2011 - 11:26 PM

Thank you bob1. In fact, I used 50ul of DNase (Roche, DNAse I recombinant) after the first wash in 450ul of cell suspension and then incubate it for 1hr in 5%CO2 and 37oC incubator. This greatly reduced the clump and I managed to get a recovery of 65-73% and viability of 74%.

#4 Dutch

Dutch

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 28 October 2011 - 11:28 AM

I've had similar issues and was actually surprised to find that there are lots of publications on the topic. The common conclusion is that faster freezing and thawing minimizes damage from ice formation/dissociation as well as hetergeneity in buffered solutions caused by different buffer components freezing and thawing at different rates. Teddytariku's claims about better viability after thawing at 37C very much mirrors what is in the literature. Bob's suggestion to freeze in liquid nitrogen is also relevant as you will get a faster freeze.

In our case, we were using more sensitive components and couldn't thaw them at 37c. We instead use a Box Scientific thaw station. It circulates ambient air over samples and allows them to cool much faster at room temp. Same concept as a convection oven I guess. The upside to using a thaw station or heat block is that the conditions are fixed. So you can proceduralize whatever works out to be the optimum condition and get more reproducible results across the board.

In the interest of good practice I'd think about developing optimized freeze thaw procedures if you don't have any. They've eliminated many of these types of head-scratchers on our end. Who knew? Good luck!





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

©1999-2013 Protocol Online, All rights reserved.