I want to detect IFNg, TNFa/IL-2 and CD107a in NK and T cells. I am going to stimulate them with different stimuli (PMA, Anti-CD16, K562, etc). Before I start my optimization I wanted to know whether overnight resting of PBMCs helps in better detection of these cytokines. The only issue of resting is that the cells die which can affect the experiment set-up. Also I don't have enough patient cells so I am bit paranoid about doing it.
Dear Nandini, I guess your question is about frozen PBMCs? (For fresh PBMCs, there would be no reason for overnight resting, in every case.) Overnight resting of thawed PBMCs is always advantageous in my experience. What happens is that dead but also dying cells are being removed during overnight incubation and subsequent washing, giving you a much cleaner and viable sample. As a result, you will get stronger but also more reliable responses. Especially dying cells can cause odd, aberrant results. Depending on the initial viability of your sample, you may see a significant reduction in the number of cells overnight, but this is the necessary sacrifice that you should make.
Dear Nandini, I guess your question is about frozen PBMCs? (For fresh PBMCs, there would be no reason for overnight resting, in every case.) Overnight resting of thawed PBMCs is always advantageous in my experience. What happens is that dead but also dying cells are being removed during overnight incubation and subsequent washing, giving you a much cleaner and viable sample. As a result, you will get stronger but also more reliable responses. Especially dying cells can cause odd, aberrant results. Depending on the initial viability of your sample, you may see a significant reduction in the number of cells overnight, but this is the necessary sacrifice that you should make.
Hi Wim, Yes my question was about frozen PBMCs. Thank you so much for your answer.
Dear Nandini
You might wish to do your own systematic evaluation of whether there is a need for overnight resting for your thawed PBMC samples before conducting your assay. This can be achieved by comparing responses obtained with fresh vs frozen/thawed samples (ie: with cells from volunteer donors), with and without prior overnight resting for the thawed samples.
You also need to be mindful of the processing upstream of your assay (PBMC isolation, freezing and thawing...) and the need to establish acceptance criteria before proceeding with the stimulation such as ie: acceptable viability post thawing, etc..
I am not sure about the assay your are using (ELIspot, ELISA, ICS) but these papers could eventually provide useful insight:
Establishing Acceptance Criteria for Cell-Mediated-Immunity Assays Using Frozen Peripheral Blood Mononuclear Cells Stored under Optimal and Suboptimal Conditions. CLINICAL AND VACCINE IMMUNOLOGY, May 2007, p. 527–537
Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization CLINICAL AND VACCINE IMMUNOLOGY, Aug. 2009, p. 1176–1186
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