Hey Elisabeth,

First If I may inquire, which cells comprise the two populations you are getting in your fresh FSC/SSC plot? I worked a while ago with other lymph nodes and got pretty much all lymphocyte (meaning one substantial population in FSC/SSC).

Second, from my experience cells, permeabilized for intracellular staining, loose features which represent the live cells, and do not appear the same in the FSC/SSC plot.

What you could do is base you're gating on fluorescent markers you already established during the fresh cells assays.

The one with the higher FSC-A but similar SSC-A (red) if you are looking for lymphocytes. I would turn the FSC slightly higher so you get a bit more separation. I was told that the group with smaller FSC are unhealthy cells, but I am not sure whether that is true or not. I found that both groups stains positive for CD3, but only the larger group stains for CD4. When you permeabilize cells, they die and the size of the cells gets smaller from the fixation process, hence there isn't such a distinct population as you see it in fresh cells. In that cause, you usually select everything that's not dead and hope that your antibodies are specific.

Hi, thanks for your advice. What i can see on these two populations is that they both comprise CD3+ CD4+, CD3+ CD8+ and B220+ lymphocytes; however in the green one (smaller in fsc) it seems there are two distinct B220+ populations : a very bright one that corresponds to the one I find in the red pop in terms of MFI, and a diminished one. Is it possible that their different size and structure relates to their activation state or something like that?

Regarding what you say, maybe I should just systematically perform a live/dead staining and gate on living cells.

Oops I think should have worded the last sentence better, the 'dead' cells after fixing are cells that were alive before fixing and are therefore smaller but still are of a reasonal size. All cells should be dead after fix, because living cells can't survive you putting holes in them so that antibodies could enter. I have found B220 tend to have 3 distinct populations... I would like to know what they are as well, let me know if you find out!

Whenever you permeabilize cells, they lose their "characteristic" FSC and SSC scatter patterns so you will not be able to distinguish them in this way as you normally do. For the PDF you've provided, the "live" cells i.e. the cells that would have been alive had you not permeabilized are the majority of the population. The only cells that are truly dead have a FSC-A and SSC-A of less than ~20. Gate on the remainder of the cells, exclude doublets, and then you'll have your cells of interest. Good luck!

In my experience (on what I can recall) with FACS using fresh mouse draining lymph node cells, I have observed two to three populations depending on their activation status: 1) dead cells and general debris due to crushing/ homogenization procedure : lowest FSC/SSC 2) lymphocytes - higher SSC 3) activated/proliferating lymphocytes - even higher SSC and also higher FSC.

With fresh cells you can discriminate dead vs. alive cells by using staining like 7AAD which stains dead cells. Of course not valuable for fixed cells (see comment of Yi Wen Qian).

With fixed cells we have also observed lower FSC/SSC and no good discrimination of populations in FSC/SSC plot possible. Thus as suggested above, use your cell markers (and appropriate isotype controls!) to identify specific staining.

I have found that the population that is low in FSC consists mainly (80-90%) of cells that take up DAPI or other Live/Dead discriminating dyes. I therefore think it advisable to just as you suggested always perform a live/dead staining (NB. with a fixable dye for intracellular staining), and gate for living cells before further analysis. Doing so almost completely "eliminates" the lower population in FSC.

I always see a shift in FSC/SSC after fix/perm. The cells change their physical properties.I always adjust the FSC/SSC voltage so that the population is draged back in the middle again. It is possible that you can see than a separation of the populations again but it does not have to be so. I would in any case recommend a live dead stain. there are good fixable life dead stains from ebioscience or invitrogen that can easily be integrated into surface staining before the fixation process. As both populations contain the cells you desire i would at first gate on both and than gate out the dead and doublets.

Of course when I said "live/dead staining" I meant a fixable viability dye that you use before the fix/perm steps. Thanks to everybody for sharing your experience, it's very nice to you all!

Hey, I was looking for the expression of a surface marker on mouse Neuro2a cells and found distinct dual populations like the ones you got. I did not permeablize them as I was looking for a surface marker. Although mine were infected with JE virus but in the uninfected ones, I did notice the trend of a dual population which is difficult to interpret why. I have a belief that cell cycle plays a role in the expression of the surface marker where during the course of the cell cycle some proteins are matured well enough to adhere to the surface where as some are just too naive; hence I get some dimly stained population of my surface marker versus some strongly stained ones. (check out the pdf). Mouse N2a is also a heterogeneous cell population from the brain of a mice and hence it could be one of the reasons to the dual population. Hence, I feel you should check about the characteristic features of your cell line and also try FACS for some surface markers which could give you an idea about the behavior of your cell line.

Hey, you can also check their size differences by preparing an smear and wright-giemsa staining. As you said, the difference may be due to their activation process which activated lymphocytes are larger. Did you treat your mice with anything or not?

Sometimes I had also the same experience after permeabilization which the cells distribution in FSC/SSC plots are better and more homogeneous than non-perm cells.

Hello Elizabeth, Your profiles look fairly standard for unfixed murine MLN.

Lower left-hand corner(black) cell debris, next, cells with compromised membranes from processing(green- these would appear dead with an appropriate stain - eg PI or fixable live/dead) followed by intact(live>90%) cells lower fsc naive than activated cell(red) overlapping with aggregates. Your fsc/ssc looks almost textbook allowing good separation with cluster discrimination, only singlets of large myelo-granulocytic may be lost from the visible screen on the ssc axis. Your problem is the result of the protocol for intracellular staining . I've used several commercial reagents and found minimal change in overall fsc/ssc - usually rbc contamination reduced by lysis. However, I have seen this with home-made reagents and pre-acquisition storage in reagents that are non-isotonic - hyperosmotic. Is the PFA iso-tonic and have you checked sample profiles BEFORE storing them in this PFA set? This is were I would suggest you look first for the source of your fsc/ssc profile collapse. Why do you need to store your cells in PFA before acquisition?

Hello Martin, thanks for your answer! The PFA in which I usually store my samples before acquisition is home-made (diluted in PBS and stored @-20°C). Maybe you're right and it affects the FSC SSC profile, that's easy to check, I will try! I often cannot access the cytometer immediately, that's why I'm used to fix before acquisition. Thanks again for your help!