Dear researchers
I'm going to do an assay for the expression levels of some cytokine and chemokine receptors on the surface of PBMCs. The PBMCs are stored in -80 0c until the experiments. does any body have an experience in this regard to share with me?
many thanks in advance for your worthy comments
Dear Azam,
i tried also to phenotype PBMC that had been stored at -80°C. Generally (and surprisingly) it worked quite well. I think it is possible to evaluate the distribution of major cell populations but I have concerns regarding the measurement of receptor densities on the cells. Moreover you may have to consider the type of freezing medium (we used Bambanker medium) and the necissity for a vital staining (with e.g. propidium Iodide or 7-AAD) to exclude false positive Events (defect cells may incorporate the antibodies and mimik a positive cell). At least a direct comparison of the immunephenotype of a PBMC sample before and after freezing would be very interesting. If I have some time left I will try this.
Best regards
Dirk Henrich
It is important that you remove all DMSO after the quick thaw of the frozen cells. Determine the ratio of viable vs dead cells. Ideally, remove dead cells by re-adhesion depending on the cell types of your interest. Propidium iodide and staining with isotype antibodies will negate false positive staining. If you are interested in a wide array of cell surface markers you will have to run pilot a time course to determine optimum antibody binding for each marker. Best wishes
I think given that such receptors can be shed, I'd spend a bit of time looking at fresh samples to make sure freezing does not cause loss of expression of any receptor of interest before starting a work programme using your frozen samples.
As Kottarappat noted, the first vital step in thawing is remove the cells from the freezing medium as quickly as possible and wash several times in warm medium (RPMI is fine). I do this by pre-warming a 50 ml tube of medium and pouring the frozen cells directly into the tube as soon as they are melted enough for the pellet to pour, pelleting the cells by centrifugation and replacing with fresh, pre-warmed media.
For some surface receptors, it can be beneficial to 'rest' the cells overnight (37 deg/complete medium), though do check that this would not cause internalisation of your receptor. This is also useful if you plan to stimulate your cells to increase cytokine and receptor expression.
A live/dead viability marker is also essential for accurate flow cytometry results, though I prefer to use one of the newer cell membrane dyes over PI or 7AAD, since they tend to bleed less between channels (I use eBiosciences fixable viability marker in efluor780) and makes panel creation easier.
Good luck with the stainings!
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