Hi Sabrina

In theory, DNAse shouldn't affect protein expression. Are you sure your DNAse is not contaminated with some protease (which will chop various proteins including receptors up)? I must tell you, the cells frozen at -80C aren't going to live for long. They all will die once thawed. It is only a matter of time. I am just curious, why do you want to digest DNA from dead cells?

Siva

Usually Proteases Like trypsin or dispase has been shown to alter the surface expression of CD4 and CD8 on T cells.The effect could to be due to protease contamination present in enzyme preparations used.Addition of serum or trypsin inhibitor might abolish this effect. Also you will find that CD3 is least affected by this enzyme treatment which has less trypsin cleavage sites that CD4 and CD8

It also worthwhile trying different clones of the antibodies against CD4 and 8 since some clones bind to epitopes that are located at sites of the antigen that are not affected by enzymatic cleavage. I agree with Asif that usage of serum can inhibit the off-target activity caused by impurities from contaminating proteases (which are most likely always present due to the process of DNase (over-)expression and subsequent purificiation.

Also, make sure to include at least 5 mM of Mg2+ (from MgCl2 for instance) and incubate at 25°C which are the optimal conditions for most efficient DNase activity. With this you can reduce your overall incubation time which in turn will be beneficial for epitope integrity and cell viability.

As a last resort and strongly depending on your downstream applications, you can of course always do an intra-cellular staining for these markers with fixation and permeabilization.

Hello,

Thanks for your answers

My intention was purify CD4 and CD8 T cells from PBMCs obtained from healthy donors frozen at -80 using the EasySep Human CD4+ (or CD8+) T Cell Enrichment Kit (StemCell Technologies). I read form the datasheet that if you are goingo to use previously frozen PBMCs, you have to incubate the cells with DNase I to eliminate the DNA from dead cells. I didn´t use the DNAse I Solution provided by stemcell, I used another but a recombinant DNAse I (Lab Roche), so I can´t expect it was contaminated with another protease.

I don't know what happened, I'll just use the enrichment kit without the DNAse treatment.

An alternative to Dnase here (which really helps prevent cell clumping) during thawing is to use Benzonase (from Millipore and others). It's rather expensive but very efficient and guaranteed free of any proteolytic activity due to its production process. I've compared Benzonase with Dnase once and they gave me the same yield, while Benzonase was a lot more gentle. I switched back to Dnase because of the price though. I'll attach for you everything I have on it, mostly found through web research (e.g. how much Mg2+ it needs eeetc). For your purpose of thawing PBMCs there is even an application note published.

I really think it's worth giving it a shot because otherwise you will lose quite many cells due to clumping. Depending on the amount of material you have this can truly make a difference. And CD4 and CD8 just are little b*tches when it comes to epitope clipping. ;)

Thank you Eric! I'll keep it in mind!

This is a very informative thread-thanks! Sabrina: have you tried benzonase as suggested by Eric? If so, has it improved your flow readouts/CD4 enrichment? 

I’m also just about to start isolating CD4Ts from thawed PBMC samples using the negative selection EasySep Human CD4+ T Cell Enrichment Kit (StemCell Technologies). The PBMC samples were frozen at approx. 10mln cells per vial (so not too many given the expected loss upon thawing) and so I’m trying to do the enrichment as efficiently as possible. 

On a slightly different note, does DNase/benzonase treatment of PBMC have any effect on the quality of downstream RNA isolation for RNAseq? I know that DNase use is mandatory during RNA isolation to remove DNA contamination, but it would be useful to know about the effects of its use upstream of RNA isolation.

Sorry Segey, I've just read this comment.. I didn't try Eric's suggestion! If you do, please, let us know.. Thanks!!!

Hi. Were there viable CD3+ cells but no presence of CD4?

That is your proof of cleavage.

DNAse should NOT cleave CD4!

perhaps Collagenase?

Hey, maybe to late to answer, but we never purify CD4+ from PBMCs straight after thawing. We always leave them to recover after thawing - at lest 6 hours (the best is to leave PBMCs over the night and next day purify CD4+). Freezing destroys surface markers, so you have to culture cells for a while so that they will express them again ;)

Thanks Mateusz- this makes sense! but will this approach be appropriate for gene expression/transcriptome analysis, since the overnight incubation might skew gene expression profiles?

Well freezing is quite a stressful process - it will definitely skew your expression. So I'd rather culture them day or two, preferably use fresh cells :P Again it will depend what kind of experiment you're doing, what answers you're looking for. I'd also check in papers what others do when they ask similar questions ;)

And for RNA isolation we use old-school TRIZOL with RNA purification columns (A&A biotechnology - RNA zol-out), it does magic in comparison to Realia kit by Promega - at least in case of CD4 T cells.