Hello all, I have a couple of queries. I am not too experienced with these methods so advice would be appreciated.
My current work requires the purification of CD4+ T Cells from mouse spleen and mesenteric lymph node. I will perform flow cytometry on cell preparations before and after CD4 selection to assess purity and the abundance of the T helper subsets. The enriched CD4 T cells will then be stimulated to produce cytokine in order to collect supernatant for a subsequent assay... I have purchased CD4 positive selection kits from Stemcell Tech (as they give better purity than negative selection and it does not matter if the CD4 binding in the positive selection activates the cells, as they will be activated anyway immediately after the purification). And for the stimulation I have purchased Dynabeads CD3/28 T-activator.
1) In a small test of this procedure I notice a high amount of debris (low forward scatter but low to mid side scatter) in the flow plots for the purified CD4 preps, that was not present in the pre-separation samples (diagram, top= crude spleen cells. Bottom= after CD4 positive selection). Could this be the beads used in the positive selection procedure? They were not expected to dissociate from the cells? I had expected the post-separation sample to produce a fairly clean plot with one major population (CD4 lymphocytes)
2) As the positive selection requires CD4 on cells to be bound with antibody and bead, does anyone know if this would interfere in any way with the CD3/28 bead binding due to proximity? Or are they quite compatible with each other?
Kind regards
Harry
1. Cell profiles change once you subject them to isolation (see below).I think what you see are dead/dying cells, not beads.
2. Usually it is fine.
Some general comments.
1. What % does S constitute in your ungated plot in the bottom (spleen post-separation)? It seems low for a sample that has undergone selection.
2. Although V shows you a super high purity (96.33%) post separation, you are grossly overestimating it since your V is within U, which is within T, which is within S, which is a small proportion of your ungated. Your V should constitute almost all of ungated for you to have a high level of purity. If you plate the cells as is, most of them are non–CD4+ cells.
3. When you are selecting/purifying cell subsets, you gate all your cells in your FSC-SSC and see what % expresses the marker you are selecting for (maybe exclude dead cells if they are few). That is the true measure of purity. If you start sub-gating, then what I mentioned above happens.
4. It seems your purification process needs optimization to keep the cells viable and also to achieve better purity. If your separation works well, you would see a single population (low SSC/FSC for lymphocytes and high FSC/SSC for monocytes) in your FSC-SSC plot and most of those cells would stain +ve.
5. Talk to a SCT rep and they can give you pointers on how to troubleshoot your separation.
To confirm that those FSC-lo/SSC-lo events in your post-separation analysis are indeed dead cells, you might consider including a stain for viability in your panel, maybe PI or one of the LIVE/DEAD stains from Invitrogen. As a rule, we do this routinely in all experiments.
N. Max Schabla is right. Including a live dead stain in your cocktail would definitely help.
1. I agree that a Live/Dead stain should always be included. But I would also be interested in knowing based on your current data, if the FSC-lo/SSC-lo events have a predominant CD4+ cell population or CD45+ or neither.
2. In general it is better to use negative isolation as a positive isolation may impact the effectiveness of downstream functional assays
The beads shouldn’t have any effect on CD4 expansion even with the bound antibody from isolation. I regularly perform magnetic isolation (positive selection) of CD8 cells and they proliferate just fine on antibody coated plates.
As for your isolation procedure, do you add FBS or BSA to your buffer solution? It’s odd that you seemingly have such high debris after isolation which could be due to your cells starving.
And I agree with Julie Joseph on your gating strategy. Particularly after isolation there should be no need to add a CD45 gate. Your purity should be 95%+ assuming the kit did its job and subgating dwindling your population size.
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