I want to do measures in separated Monocytes and Lymphocytes derived from PBMCs but my separation protocol lasts about an hour and I need to use my group (n=14) in one experiment, so I could not do it in a day. Instead I would like to separate the cells and freezee them in the working concentration until the experiment in order to thaw them all at once and perform the experiment.
The issue is that is a cell death experiment and I don't know how much it would affect them.
I am currrently making a standarization but I would like your suggestions
Thanks
Freezing/Thawing isolated monocytes without major cell loss is almost impossible, due to the intrinsic fragility of these cells (this is less of a problem for lymphocytes). For this reason you cannot freeze them at the working concentration and expect getting the same number of cells before freezing and after thawing. And even if you freeze an excessive number of cells to have enough cells following thawing, you will probably wonder what is the nature of the cells that are not here anymore and to which extent your experiment reflects anything physiologic. To make it short, freezing isolated monocytes does not work.
Of course this depends on the exact nature of the experiment you plan to do, and it would help if you shared more details.ds notably on the quality of the serum you use and the quality of your freezing/thawing procedure). However, in your case freezing PBMCs could be interesting if you need to make your experiment in several steps and correct for a batch effect. In an extreme situation, rather than performing your experiment on 14 samples at once (which would be technically difficult), you could run your experiments on 2 samples per day for 7 days and add each day one common sample (one out of 7 cryotubes from the same PBMCs) to correct for the batch-effect. But maybe this is what you meant by standardization.
But of course the best solution depends on the exact nature of the experiment you plan to do, and it would help if you shared more details.
Julien Verdier is right. Isolated cell subsets do not do well once frozen. A significant proportion of cells do not survive freeze-thaw even when using optimized free-thaw protocols. Monocytes constitute
Thank you so much Julie Joseph and Julien Verdier I'm actually going to do exactly as you suggest and make 4 batches of 7 each (I have controls and patients). For me is very important to have the cells as little perturbed as I can in order not to modify the phenotype.
My goal is to test sensitivity to TRAIL and FasL in monocytes and lymphocytes of my subjects, I'll separate with mojosort (bead assay), plate them and use recombinant ligands to induce apoptosis and measure it with Caspase Glo (Caspases 3, 7 and 8). I actually got a second magnet (mojosort gives you a single magnet but they lend me another) so I can make the 7 separations in a day and put the recombinant ligands at noon, It's going to be long but I'll work.
Thank you so much! :D