Whole blood is better to study than isolated cells because the cell isolation and harvesting will be variable, before you even get to freezing and thawing, so by using whole blood you see what is actually there in totality. After staining we lyse and fix (BD FACSLyse and BD CELLFix) which gives you a stained cell prep which you can keep in the dark at 4C for a couple of days before running the flow cytometer. In around 20 years of running research flow cytometry we have never seen instrument drift (unless something major happens like LASER failure) so you do not need to set up your instrument daily. That is for clinical labs who need to provide validated daily quality assurance that their instruments are working properly. For prolonging the life of samples you could look at preservatives such as Streck Cell Preservative, so that you could arrange to run multiple samples simultaneously (which I admit is easier than running many single samples on different days - depending on how many tubes are in your assay).

It is also possible to freeze whole blood (just add DMSO to 10%) - the lymphocytes in that survive well. Only lymphocytes and lymphocyte-like rare cells such as HSPC will survive freezing well anyway - other leukocytes will not - whether or not you isolate mononuclears or leave them in whole blood. That would avoid the variability introduced by the mononuclear buoyant-density isolation (and save you some time).

Yes u can do it as dfescribed by john, but you will loose sensitivity due to freeze-thawing. Take care

I am not sure about chemokine receptors but CD4 and CD8 in our hands are detected at same percentage before and after cryopreservation. All you need is a reliable and validated cryopreservation protocol. In our protocol, we use a combination of hydroxyethyl starch and DMSO (7.5% from each) in colorless RPMI 1640.

After thawing at 37 degrees we leave the cells in fresh RPMI 1640 containing 10% FBS overnight in +4 C. Then we do stainings in following morning. You need to validate this or any other protocol in your lab and see what works best in your hands.

Once you optimize the protocol you may use it for different markers.

Best wishes,

I do not have real experience on this specific sorted cess subtype but on general point of view analysis works well even after a stimulation. Anyway, it is recommanded to keep the cell in the incubator for a few hours in culture medium to give them the chance to recover before any treatment (stimulation and /or labelling).

Can I ask why you plan to freeze your cells? Is it because you plan to look at some change over time and you want to collect over time then analyse simultaneously? Why not just measure fresh at each time point? If you set aside your staining (antibody) panel and reserve it just for this study, there is probably much less variability in the staining technique repetition than in the mononuclear cell isolation / freezing / thawing, especially if you stain on fresh whole blood, not MNC. All of the above is good advice - but you can see the sources of possible variation if you freeze/thaw - and also if you isolate mononuclear cells from whole blood. Also I guess you may be interested in proportions of cells rather than absolute brightness of staining (MFI), so it might not even be that critical if your antibody batch changed (but I would try to keep the same batch - or be prepared to convince a referee why it wasn't important)

The reason why I want to freeze the cells is because I can only include 1 patients pr. day. Then I need to setup the flowcytometer each day and run 1 sample and then shut it down again. Then in 1 or 2 weeks I'll included the next one and so forth.

My control group is easier as we have a lot of "normal" people running around our science facility as we are on Campus (University of Southern Denmark).

Would you recommend that I use whole blood and a lysis buffer?

I get 18mL of whole blood from each patient/control. I'm using the buffy coat to do a magnetic bead separation in different subtypes that I analyse for different K-channels. That's the reason why I was planning on doing the flowcytometry from the buffy coat. But It's not that hard just to take a few mL to do the flowcytometer analysis afterwards.

Is it enough just to compare the fresh PBMCs vs. frozen PBMCs from one patient and if the relative distribution doesn't differ - we might as well freeze it... We are only planning on having 7-10 persons in each group, so the pilot study will be close to the "larger scale" study :)

I did several trials with Frozen cells and it not working very weel compare to fresh cells in Flow cytometry.

Whole blood is better to study than isolated cells because the cell isolation and harvesting will be variable, before you even get to freezing and thawing, so by using whole blood you see what is actually there in totality. After staining we lyse and fix (BD FACSLyse and BD CELLFix) which gives you a stained cell prep which you can keep in the dark at 4C for a couple of days before running the flow cytometer. In around 20 years of running research flow cytometry we have never seen instrument drift (unless something major happens like LASER failure) so you do not need to set up your instrument daily. That is for clinical labs who need to provide validated daily quality assurance that their instruments are working properly. For prolonging the life of samples you could look at preservatives such as Streck Cell Preservative, so that you could arrange to run multiple samples simultaneously (which I admit is easier than running many single samples on different days - depending on how many tubes are in your assay).

It is also possible to freeze whole blood (just add DMSO to 10%) - the lymphocytes in that survive well. Only lymphocytes and lymphocyte-like rare cells such as HSPC will survive freezing well anyway - other leukocytes will not - whether or not you isolate mononuclears or leave them in whole blood. That would avoid the variability introduced by the mononuclear buoyant-density isolation (and save you some time).

I agree with George Barclay. I have tested the cytokine profile between fresh and frozen PBMC's and found a 10% reduction in the frozen PBMC's. Keep this in mind when you are setting up your experiments.

I wouldn´t froze cells, viability diminishes a lot..

Hi Chandra. Of Course I will. I've decided to do the flowcytometri analyses on freshly isolated cells. I will use a lysis buffer to get rid of the RBCs. Then I'll include a CD3 (overall t-cell marker) antibody and then use CD4/8/CCR7.

This should give the best result and I am probably going to do as suggested by George Barclay regarding the CellFix and Streck Cell preservative, as this would be a lot easier than running each sample on separate days (maybe two samples/day)...

The frozen cells is out of the question for now :-) I'll let you know when the experiment has been done.

Best

George Barclay, I have the same problem and I cant manage to do fcytometry even once a week cause I dont know how long it takes to find new patients. another problem is I am looking for  ICAM-1 (which expresses on monocyte)? and as you said just lymphocytes wiil be survived in freezing protocol. fresh sample is the only way?