It could be doublets, that is, two cells stuck together that go through the laser at the same time giving the appearance of a cell with twice as much of everything.

For your initial gating, plot FSC-A against FSC-W.

You should get something like the attached figure.

Gate on singlets.

In my experience PMA/iono stimulus changes granularity and induces potent down-regulation of CD4 influencing the FSC and SSC ! You could try PHA, where there is no change in FS vs SS but induces a lots of blasts.

Thanks for the response Aditya. This might explain it. One of the 2 populations is higher on SSC, but they are comparable on FSC.

Are they still alive? Your dead/apoptotic cells will usually shift on FSC/SSC (FSC goes down, SSC goes up usually)

They are fixed cells. Both populations are the same size, different SSC. Dead cells are very clearly smaller than both. Thanks

If the experiment is done in an appropriate condition ruling out all other flaws (Dead cells etc,), PMA or PHA treated cells increase in granularity reflecting on shift in SSC. It seems all cells were not activated evenly, hence only activated cells are showing the shift. You have not given sufficient information. So cannot comment more without seeing the data. Were the cells stained for lymphocyte-specific markers?

PBMCs were treated with PMA/Ionomycin for 4 hours and then fixed in PFA. PBMCs were intracellularly stained for IFNg a week later. I haven't used any lymphocyte-specific markers. I'd like to look at changes in IFNg expression in general lymphocyte population, but have 2 very distinct populations.

Maybe it would be worth to stain for lymphocyte specific markers, so that you can assess if this shift is widespread among different cell populations, or just specific for ones. I've never directly stimulated PBMCs, but with purified CD4+ cells, I do observe the shift in FCS/SSC blots upon activation, as Aditya commented.

Just by curiosity, is it possible for you to stain earlier? I have been recommended to wait no more than 48 h between fixation with PFA and staining. I've never tried to do it later, I am not sure if that could somehow affect your samples.

It could be doublets, that is, two cells stuck together that go through the laser at the same time giving the appearance of a cell with twice as much of everything.

For your initial gating, plot FSC-A against FSC-W.

You should get something like the attached figure.

Gate on singlets.

In doublets, there is exactly double the value of SSC and FSC in the neighbouring population and the cells shift diagonally. It can be differentiated from singlet easily as shown by Pedro Velica. You may also encounter monocytes near lymphocytes and doublets. It is better to stain lymphocyte population to avoid confusions. Stain surface markers before fixing the cells.

Perhaps different states of cell activation, cells change in size and granularity with activation therefore affecting forward and side scatter

try running samples at the completion of the assay, rather than fixing and storing

look for expression of different activation markers

I ruled out doublets since they have only increased on SSC, and as Anbazhagan has pointed out, they should shift on FSC too. I have acquired the data on a Calibur which has no FSC - W parameter unfortunately.

In your case, it's not a doublet issue. It also could be b-cell and t-cell population as they may show different granularity levels up on PMA treatment. Check for cd19 and cd4.

The PBMCs are being cultured in conditioned media for 72h before addition of PFA/Ionomycin. A PFA/Ionomycin/Brefeldin A cocktail is added directly to culture with gentle mixing, as opposed to re-suspension. Maybe, as Dominic suggests, I don't have a single cell suspension for stimulation and it's generating odd FSC/SSC profiles.

The population with higher FSC is probably activated cells, if you stain for CD45RO expression , theses will probably all be positive, whereas only a proportion in the lower FSC population may express CD45RO.

cheers

kay

Increases in side scatter are proportional to the granularity of the cell. As Kay mentioned, activation is the most likely cause for this increase in granularity. The granularity increase is typically NOT proportional to increases in forward scatter, thus the cells would not be on a diagonal, as seen in doublets.

I am sure you are doing it already, but you should always strain/filter your cells before running them on flow (especially if they have been sitting for so long - 1 week is a LONG time to wait). I would recommend the 40uM cell strainers (https://us.vwr.com/store/catalog/product.jsp?product_id=4643994) . A typical cytometry SIP(sample injection port - long silver needle thingy) has a 100uM bored hole. So if you are also having clogging issues,doublets are a definite problem you should be considering. With a 40uM strainer you could still get doublets, but it will take care of those larger aggregates and a single cell populations are much more likely. I typically filter ~200ul through multiple areas of the mesh (8-10 per filter) using the bottom, sides, etc. to make it more economical. Unless you have money to burn, don't buy the FACs tube cap filters. Get the big ones for 50ml conicals.

http://www.thomassci.com/_resources/_global/media/resized/00006/ihwx.5c09c1e5-a139-4111-8164-d85604a6879b.500.500.jpg

https://us.vwr.com/store/catalog/product.jsp?product_id=4643994

Thanks David