I did an timecourse assay and I noticed that particularly in my unstimulated controls I see two populations (see attached figure). When I gate on them they both seem to express CD3. These are PBMCs (previously frozen) incubated in cRPMI. I expected the lymphocyte population to appear approximately where the population on the left is. When I stimulate with CD3/CD28 beads I see that the population to the right seem disappear. Can anyone explain what kind of cells this are?
Dear Dr Håvard Foyn, these cells may be 1) blast cells; 2) cellular aggregates of 2 or more cells. You can dissect both on FSC-A/FSC-H plots; 3) these may be dead cells that acquire mAbs non-specifically. In this case you can exclude them from analysis by PI staining.
Dear Dr Håvard Foyn, these cells may be 1) blast cells; 2) cellular aggregates of 2 or more cells. You can dissect both on FSC-A/FSC-H plots; 3) these may be dead cells that acquire mAbs non-specifically. In this case you can exclude them from analysis by PI staining.
Thanks for your reply Dimitri. I left out the FSC-A/FSC-H plots, but they indicate that they are all single cells (attached: All cells top, bottom is gated on population to the left and right). I have unfortunately not included a viability dye (will do that from now on:) ), so they might be dead cells, but I would expect dead cells to have lower FSC-A.
Fine! Lower FSC-A is characteristic for cells dying by apoptosis in in vitro cultures. Recently frozen cells is different matter.
Hello
Yes , you can start with the negative selection of these unwanted events. As a general guide, that the dead/apoptotic cells will usually shift on FSC/SSC (FSC goes down, SSC goes up usually) .
Good Luck
Hi,
as Saleh stated, dead/dying cells should be completely elsewhere on the plot, but since they are not doublets (have spherical shape by FSC-H/FSC-A) and have almost twice the size (FSC-A) are they not by any chance proliferating? But then you should see a small number of doublets on FSC-H/FSC-A (after clefting, prior to division), unless a G2 arrest occurs...CD3/CD28 stimulates differentiation or proliferation? If the former, that would support the proliferation hypothesis...
Could you please add PI or DAPI to exclude that the population on the left are not dead cells? How long have you stimulated your cells with CD3/CD28?
Raffaello is right about the stimulation length - If you use Dynabeads for stimulation and expansion, they state in step 5: "Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures.", which could further support the hypothesis, that the rightmost population is proliferating T cells (which are almost 100% CD3+ in your plot) - if the stimulation proceeds for several days...
Hello,
I agree with what Rafaello and Jan. You might be also having exhausting issues. To contrast, you could either lower the stim or try alternatives like e.g. plate-bound CD3e and soluble anti-CD28.
As you are recovering these cells from frozen PBMCs, I would expect a significant proportion of dead cells. Are those plots before or after stimulation? For how long is your culture going? It would useful to see the CD3/28 stimulated plots. No matter what, adding an apoptotic cell marker would be great, but the cells on the right definitely have the characteristics of blasting CD3 cells.
Typical picture of an uncorrectly frozen cell sample. Something went wrong during freezing/defreezing procedure.
Thanks for all your replies. I have been a little busy, but have now also included a viability dye (fixable viability stain 700 from BD). So this is a new run with the same frozen cells. And both population are viable (have only shown one picture, but it looks the same for both populations). The funny thing is that in this run I see the population in both stimulated (with dynabeads coated with CD3/28 antibody for 5 hours) and unstimulated samples (no beads).
I was at a BD flow course and and had the oppertunity to ask one of the people there. Her response was: "Oh, the peanut!". She had seen it in mice cells, but not in human cells and she did not know why there were two populations.
Do you have any strategy in place to exclude cell aggregates (FSC-H vs FSC-W or FCS-A vs FSC-H)? I think that the gated 1.03 and 5.04 populations are cell aggregates that need to be removed from your analysis.
Yes, I did not show it, but I gated on FSC-A vs FSC-H. They are single cells.
Could you please share a couple of representative FCS files with google drive? Which instrument have you used to run your samples?
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