Hi Alberta, we also ficoll and freeze our PBMC. There should be no problem for subsequent Foxp3 staining. Which protocols do you need?

Yes, we permorfmed ICFC in frozen cells. What is important, you must have good banking protocol, check the viabiliy after thawing and with you can gate out dead cells e.g. with PI staining. There is question shall wee rest the cells after thawing or not.You can fin papers pro and contra for overnight resting. For sure it helps to eliminate early apoptotic cells, which will become dead.

You could try methanol fixation at -20C. It would permeabilise and fix them, with possible storage for several months I believe.

For intracelllar cytokines they probably need live cells for stimulations and incubation with berfeldin etc, so I do not advise fixing with metanol.

Fair point.

With a protocol that involves stimulation-freezing-thawing-staining, you run the risk of both protein degradation and differential protein expression induced by the freeze-thaw cycle and the culture time pre and post freeze.

Is there any reason why you can't isolate the cells, freeze them and do the stimulation after thawing? You may get cleaner results this way.

Also, we have broken up the the protocol for permiablizing using BD's Perm-wash into two days, with only 45min-1h being spent on the first day and the rest of the staining time being spent on the second day. If you need a protocol for that, let me know.

We did most of our assays using thawed PBMC (to synchronize samples and assays). We used eBioscience Treg Foxp3 kit. You can check out our recent paper: Klein eji p533 2013 flt3 expands human Treg

You will notice in Weisan's protocol that they manipulated frozen cells rather than trying to stain after a freeze-thaw cycle on stimulated cells.

Yes , you can freeze PBMC in the freezing medium (RPMI, FCS, DMSO) .

We did not see significant difference when staining for Foxp3 before and after freezing the cells. Eliminating dead cells after thawing cells and before staining for Foxp3 may not be a good idea. If some Foxp3+ cells died during freezing it does not mean that particular cell was not Foxp3+ cell ! Foxp3 is an intranuclear protein This could be different when staining for cytokines, since inmany ocasions celles need to be stimulated prior staining. In principle, eliminating dead cells before staining would artificially reduce the number of positive cells either for Foxp3 or any other cytokine.

I would like to add that if your are interested in looking at the percentage of these cell population (Tregs and GD) you don't require to stimulate the cells for intracellular cytokines. Just after separation fix cells with 1% paraformaldehyde and then permeabilize with saponin buffer. Then add cocktail of antobodies including intracellular ones.

You're right. Only if you need to look at the percent of inducible cells secreting a particular cytokine. Foxp3 can also be induced by in vitro stimulation with CD3/CD28 Abs plus TGF beta. It depends on what you're looking at.

Yes, you can.

Thank you all for your answers, I might have not been clear though..I isolate PBMCs from human peripheral blood by Ficoll-paque and then want to freeze them until the assay which be 4 hrs stimulation with protein transport inhibitor then followed by surface staining and then İCC cytokine and Foxp3 staining. The live dead stain from invitrogen would be to exclude any false positive cytokine+Foxp3+ and this can be done on fixed permeabilized cells.

Hi Alberta. Freeze-thawing PBMC for subsequent FOXP3 staining won't be a problem. In our experience, the highest CD25/FOXP3 expressing cells (population II as defined by Miyara et al) are a little fragile and don't survive too well after defrosting, so if your plan is to look at sub-populations of Tregs you might want to avoid freeze-thawing. Otherwise, you should be just fine.

Incidentally, if your time from separation to FOXP3 assay is pretty short, you might be able to avoid freeze-thawing by keeping the cells in some culture medium +/- small amount of IL-2 (depending on length of time) in an incubator.

Hope this helps.

You can freeze PBMC after Ficoll separation.

This article may be of help

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3328586/

By gating out dead cells, I also didn't notice an appreciable differences when comparing fresh PBMC vs. frozen from the same donor (same blood draw). Some people are concerned that Treg activity is decreased after being frozen. I didn't notice this while staining for pSTAT5 using Phosflow Buffers from BD. I would recommend using a reliable clone.

Additionally if the cells are thawed into media that contains benzonase (a nucleic acid digestion enzyme), I've noticed mucch cleaner stains and lower backgrouns upon stimulation. It's an expensive reagent, though, and DNase would serve you almost as well.

Good luck!

I recommend performing the activation assay prior to freezing.

You can certainly freeze PBMCs for subsequent stimulation; the cells generally require a brief rest in media after thawing before regaining full functionality, and DNase or benzonase are helpful in preventing clumping due to DNA released from dead cells.

That being said, I agree with Luise. I prefer to perform my stimulations on freshly isolated PBMCs (or more frequently whole blood) and then fix the cells. The fixed cells can then be frozen and thawed for IC staining without having to worry about potential changes in viability or functionality.

Hi Alberta,

You certainly can detect Foxp3 and cytokine producing cells using frozen PBMC.

We use Brefeldin-A (Golgiplug) from BD Bioscience http://www.bd.com/resource.aspx?IDX=17721 during culture

and the BD Foxp3 Buffer Set (Cat no 560098).

From experience:

IFNg, TNF and IL-2 are easy to detect.

IL-10 is harder and you may need to optimise (e.g. culture length)

Thank you all...Adeeb and Melissa what is the concentration of DNAse I should use and how long should the cells rest before activation?

For Behdad: I want to discriminate if certain Treg populations are more prone to secreting IL-17A or IL-10 as in Raffin, JI 2011 they state that a certain population express high levels of IL-1R and I am looking in patients that have increased IL-1b production. This is the central memory population ..would I lose them?

Hi Alberta. We've just published that data in EJI. Have a look at the paper. The population you want is population III expressing CD161. IL-1R1 is upregulated by IL-2, so Tregs generated during exposure to IL-2 might have higher IL-1R1 expression than non-Tregs. You would probably want to compare like with like (pop III CD161- vs pop III 161+). These cells should survive freeze-thawing. Let me know if you need to know anything else.

Dear Behdad, I sent you an message but was able to get the proof of the paper. Very nice paper very nice work. Sent more details and questions in the message.

Adeeb, you said that you have stained frozen, fixed cells. Do you have/know of any publications using this method?

Christopher, I should clarify that I generally do my stimulation and surface staining in whole blood, then add BD FACSLyse and then freeze the fixed lysed pellet for subsequent intracellular staining. Holden Maecker's group has published a few publications validating the method. Here is an example:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1184077/

BMC Immunol. 2005 Jun 24;6:13.

Standardization of cytokine flow cytometry assays.

Maecker HT, Rinfret A, D'Souza P, Darden J, Roig E, Landry C, Hayes P, Birungi J, Anzala O, Garcia M, Harari A, Frank I, Baydo R, Baker M, Holbrook J, Ottinger J, Lamoreaux L, Epling CL, Sinclair E, Suni MA, Punt K, Calarota S, El-Bahi S, Alter G, Maila H, Kuta E, Cox J, Gray C, Altfeld M, Nougarede N, Boyer J, Tussey L, Tobery T, Bredt B, Roederer M, Koup R, Maino VC, Weinhold K, Pantaleo G, Gilmour J, Horton H, Sekaly RP.

I've found that some cell surface epitopes aren't preserved following fixation and freezing so I tend to do my surface stains prior to this. That being said, there are studies that have compared some surface stains before and after various permutations of fixation and freezing and have found minimal differences, e.g.,

http://onlinelibrary.wiley.com/doi/10.1002/cyto.b.20038/full

Cytometry B Clin Cytom. 2005 Jan;63(1):47-55.

Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry.

Pinto LA, Trivett MT, Wallace D, Higgins J, Baseler M, Terabe M, Belyakov IM, Berzofsky JA, Hildesheim A.

I think it really depends on the antibody clones you use. eBioscience has a helpful listing of the relative performance of a number of antibody clones before and after fixation:

http://www.ebioscience.com/resources/application/flow-cytometry/clone-performance-after-fix-perm.htm

Alberta, I generally use DNAse at a concentration of 0.1mg/ml when thawing PBMCs.

In my experince it works fine and usually result in no differences compare with freshly drawn blood