I want to perform flow cytometric analysis of T reg cell (Both nTregs and Tr1) and CD4 and CD8 cell in PBMC's from patient blood samples. Can I perform the cellular analysis simultaneously? Please suggest the procedure. Also as I was looking through various research articals they isolate T reg cell and then perform the flow cytometry based analysis.
I have cryopreserved PBMC's. Please suggest.
I think you will have difficulties on cryopreserved PBMC for Treg analysis. Accurate CD25 measurements may be difficult. I suggest to keep your cells in refrigerator over night after thawing and make a validation study with the fresh samples before running your samples. Please read the Finak et al. article "Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium" Scientific Reports 6, Article number: 20686 (2016) which is a good guide.
Best wishes,
Thank you Gulderen Yanikkaya Demirel. Can I measure number of CD4/ CD8 and T reg cells (CD4. CD25 and Foxp3) simultaneously in single experiment by labelling with different fluorescent markers.
You may choose to use already validated commercial antibody combinations or buy single conjugated antibodies to make your own antibody cocktail. Since Foxp3 is an intracellular protein, you need to make a surface and intracellular staining validation. I suggest to use a commercial cell permeabilisation/fixation kit. If you do not have experience in intracellular staining, first validate your intracellular staining.
For the Tregs, You may use a five color panel CD3/CD4/CD8/CD127/Foxp3. We prepare three tubes: 1. tube: Unstained cells 2.tube: Surface markers CD3/CD4/CD8/CD127 3. tube: Surface markers plus Foxp3. With this staining you will be able to determine CD4+CD25+CD127negFoxp+ and CD8+CD25+CD127negFoxp+ cells. CD127 is used for determining T effector cells.
Hi Lovlesh,
Gulderen has given you perfect advice. I might just add that frozen samples have reduced FoxP3 expression (I have tested it myself) compared to their fresh counterparts. Furthermore, FoxP3 expression varies widely in humans, so don't be discouraged if you don't see FoxP3 in many of your samples.
To isolate TRegs, you cannot use FoxP3 (as FoxP3 is an intracellular marker and you need to fix/kill cells in order to stain something intracellularly). I have seen people just use CD3+CD4+CD25+CD127- for isolation (are you going to use flow sorting for isolation?). However, CD25 expression will be completely lost in your frozen samples. Like Gulderen suggested, you may have to incubate your cells overnight after reviving, preferably in the presence of IL-2 to up regulate CD25 expression.
And just incase it wasn't obvious, you CAN simultaneously stain for CD4/CD8/FoxP3 in your samples. I would advise using a very bright fluorophore for FoxP3 antibody such as AF647 or BV421. Also, be careful using the right buffers for fixation/permeabilization as not all antibodies are compatible with all fix/perm buffers. BD has a very very good resource for figuring that stuff out. Here's the link:
https://www.cytobank.org/facselect/
I hope this helps.
Good luck!
eBioscience have good FoxP3 staining kits (fixation, fix/permeabilization buffers, antibodies: http://www.ebioscience.com/human-foxp3-antibody-pe-set-236a-e7-set.htm. If you are going to use other fix/ perm buffers make sure you permeabilize cells well enough to permeabilize nuclear membrane, not only surface membrane.
I used eBio FoxP3 buffers for many NUCLEAR proteins in IF and flow cytometry for mouse and human cells and they work great (see picture of single LnCAP cell nucleus stained for androgen receptor as my profile picture as an example). I will recommend to stain surface proteins first, then do fixation, permeabilization and staining for intracellular FoxP3. I will also recommend to do fixable viability (Zombie dyes, Aqua dead dyes, etc) staining even before surface staining and fixation/permeabilization step to remove dead (before fixation ) cells from analysis later, especially for frozen PBMCs. If you use fresh PBMCs their viability after Ficoll isolation should be good enough to skip fixable viability staining, but frozen PBMCs will give you many dead cells.
The number of colors/dyes used for flow cytometry simultaneously depends on lasers/filters availability, so check your flow cytometry machine first. And use the brightest color possible based on fluorochrome stain index for FoxP3 and CD25 as already mentioned by other posts. Link to stain index:
https://www.bdbiosciences.com/documents/Fluorochrome-Chart-Relative-Brightness.pdf
By the way in culture all mouse naive CD4 T-cells that are CD25- become CD25+ within couple of divisions. We've tested this in the lab with annexin V, CFSE, CD4 and CD25 staining long ago. Only dead/apoptotic and some undivided cells look as CD25- in cultures of magnetically isolated mouse CD4 T cells in presence of irradiated APCs . I've never done this with human cells, but the pattern of CD25 expression could be the same. So you might culture your cells first in presence of antigen presenting cells or CD3/CD28 to get more Tregs, depending on aims of the experiment.
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