Of these three plots, the one with the smallest gate (the top) is probably the best. Resting lymphocytes are relatively small (FSC-low/int), and their relatively smooth membranes and lack of granules make them SSC-low. Since parameters such as staining brightness and amount of scatter are ultimately determined by the configuration of your instrument (i.e., laser power, PMT voltage), you can really only identify populations based on their characteristics relative to other populations in your sample - so in that sense there is some subjectivity involved. It looks like you have two populations in your scatter plots - my guess would be that the larger population are monocytes, and the smaller are lymphocytes, so you may want to adjust your gating to exclude larger cells. Do you have any specific markers that you're going to stain these cells with? That might help identify them with more certainty. 

It is dependent on what are you trying to find out. What do you want to determine? Percentage of T cells, B cells, specific subsets of lymphocytes, expression of cell surface or intracellular antigens?

BTW, why did you use ficoll? there are not many granulocytes, and they can be easily gated out using Ly6G

Of these three plots, the one with the smallest gate (the top) is probably the best. Resting lymphocytes are relatively small (FSC-low/int), and their relatively smooth membranes and lack of granules make them SSC-low. Since parameters such as staining brightness and amount of scatter are ultimately determined by the configuration of your instrument (i.e., laser power, PMT voltage), you can really only identify populations based on their characteristics relative to other populations in your sample - so in that sense there is some subjectivity involved. It looks like you have two populations in your scatter plots - my guess would be that the larger population are monocytes, and the smaller are lymphocytes, so you may want to adjust your gating to exclude larger cells. Do you have any specific markers that you're going to stain these cells with? That might help identify them with more certainty. 

@Sergey Ryzhov @N. Max Schabla Thank you for your replies. I want to determine the proportion of Th1 cells in mouse spleen, ie CD3+CD8-gammaIFN+ cells. I stain CD3 CD8 and gammma-IFN, and when I include the two populations, they both include CD3+ CD8- cells, here are my results,so I wonder if both of them are T lymphocytes.And the question about why I use Ficoll, I am really sorry that I don't know,but I will try to figure it  out.

Okay. If you already using CD3 then you don't need to gate lymphocytes on SSC/FSC plot. 

1) I prefer to analyze all cells simply excluding very low FSC events(both high and low SSC, please see dot plot, gate R1). These events are erytrocytes, cell debris, apoptotic cells. 

2) Make sure that you analyzing viable cells (gate R2) and singlets (gate R3).

3) Exlude CD8 positive cells like you did it already

4) Create CD3/IFNgamma plot using CD3posCD8neg population and determine percent of CD3posIFNpos cells. 

Alternatively, you can use CD4 antibody instead of CD8, or in combination with CD8 (if you using cytometer with more then one laser). This aproach is a little bit more straightforward if you want to analyze Th1 cells only. 

5) Then present the data as % of total splenocytes (don't forget to make adjustment for excluded CD8 cells) and number (calculated from total number of cells) of CD3posIFNpos cells.

One question with you current flow is that you found too high percentage of CD3 positive cells (more then 70% in "lymphocytes" gate). Usually, there is 40% to 60% of B cells (CD19 pos) and 30% to 50% of T cells (CD3) in C57Bl6, FVB and BalB/c mice. This could come from your Ficoll purification step which is not necessary here. 

-Sergey

Hello, I'm just curious if you were able to run acquire data of your crude splenocyte samples that were not run with Ficoll on the same experiment with the samples you have after cell separation using Ficoll, then compare their FSC vs SSC plots to have a good look on the difference of between non-Ficoll and Ficoll-processed splenocytes?

Then backgate all the markers you used for your B cells, T cells, etc to the FSC vs SSC plots so you would have an idea where all these populations can be gated as your reference. I was using a FlowCytometry Simulator when I started learning about Flow Cytometry and one of the examples shown was the difference of your population profiles between crude cells and cells separated by density gradient.

I hope this will still help, all the answers given are really helpful. I also ought to run splenocytes in the next few days.

Sincerely,

John

Dear Sergey

Thank you so much for your very helpful suggestions.I still have a few questions to ask.

1)  How did you isolate lymphocytes from splenocytes？

2) Why the percentage of CD3 positive cells would get higher after I used  Ficoll purification step?

And about the CD4, because I use PMA+ionomycin+BFA to stimulate the lymphoctes before stain antibodies, so I choose CD3 and CD8.

Hua.

Dear John

Thank you very much for your reply.I will give it a try to see the difference between these two methods. It is a very useful suggestion.

Hua 

1) I did use LIVE/DEAD® Fixable  violent dye from ThermoFisher/Invitrogen. This is an amino-reactive fluorescent dye.  These dyes available also from BioLegend (Zombie ) and Affymetrix/eBioscience (Fixable viability).  You will need to incubate cells with viability dye after stimulation of cells and before fixation. 

2) This is not nessesary to isolate lymphocytes for the measurement of IFNg production by spleenic T cells, unless you study interaction between myeloid and lymphoid cells. It requires, however, slightly different study design. Everything you need to do is just gate CD3 or CD3/CD4 cells.

3) That was my suggestion based on high level of CD3 lymphocytes in your study. However, I might be wrong, e.g. I don't know how long did you stimulate cells. It is just not nessesary to furhter purify MNC from spleen because it is already 85-90%% of them. Try to keep it simple! Every additional procedure which is not nessesary brings  some uncertainty. What are you trying to reach removing 5%-10% of granulocytes? Do you work with tumor-bearing mice? The percent of MNC cells is less then 80% in spleen of your experimental mice?

4) If you can see CD8 after PMA stimulation you will find  CD4 expression. Couple tips to get better CD4/CD8 staining. Add BFA at the same time with PMA/iono. Do not stimulate longer then 3-4h. I am doing CD4/CD8 stainig twice 1) after 3h stimulation with PMA/iono/BFA and before fixation, and 2) after permeabilization, during incubation with anti-IFNg Abs.

-Sergey