In theory, any type of sample can be used for flow cytometry as all that is required is a single cell suspension of intact cellular structures. We routinely run various immune preparations, and they can be performed both fresh (when performing measurements that require live cells like redox or metabolism) or fixed (when performing immunophenotyping). The frozen samples would first have to be thawed before running them on flow, but as long as the cells are intact there should not be a problem. It appears you want to perform immunophenotyping, so you could thaw the cells, stain them with respective antibodies, and then fix them before analysis.

As far as the whole blood vs plasma question, you will need whole blood to examine cells. If you are looking for whole cells, then you cannot perform flow on plasma as by definition it is a cell free liquid. The RBC's in whole blood can sometimes interfere with analysis because they are in such high abundance, so we and others often perform a RBC lysis stage (or a Ficoll gradient) to remove these and analyze the remaining white blood cells only.

Hopefully this helps, and good luck.

~Adam

In theory, any type of sample can be used for flow cytometry as all that is required is a single cell suspension of intact cellular structures. We routinely run various immune preparations, and they can be performed both fresh (when performing measurements that require live cells like redox or metabolism) or fixed (when performing immunophenotyping). The frozen samples would first have to be thawed before running them on flow, but as long as the cells are intact there should not be a problem. It appears you want to perform immunophenotyping, so you could thaw the cells, stain them with respective antibodies, and then fix them before analysis.

As far as the whole blood vs plasma question, you will need whole blood to examine cells. If you are looking for whole cells, then you cannot perform flow on plasma as by definition it is a cell free liquid. The RBC's in whole blood can sometimes interfere with analysis because they are in such high abundance, so we and others often perform a RBC lysis stage (or a Ficoll gradient) to remove these and analyze the remaining white blood cells only.

Hopefully this helps, and good luck.

~Adam

Dear Philip, in FACS analysis use freshly isolated mononuclear cells. Any storing procedures, such as freezing, fixation, etc. will negatively affect the quality of subsequent analysis. Use these procedures only if this is absolutely required. In this case you will need to perform big preliminary work to determine working combination of mAbs and storing procedures that minimally affects results.

If you are also interested in granulocytes, you have to use fresh blood, since they don't survive freezing. An RBC lysis step is also required to get good results. From your question it is not quite clear what exactly your frozen samples look like. If they are mononuclear cells frozen with DMSO etc. you can still do FACS analysis (although I agree with Dmitry that there are some problems associated with that). Whole blood frozen down without any cyropreseration agent such as DMSO are not suitable since the cells are likely to be dead. Also, I would avoid comparing fresh and frozen samples.

Hope that helps.

Hi Philip,

In addition to the points above I would suggest adding a viability discriminator (such as propidium iodide or similar) when using cells which have been cryopreserved or lifted from a flask. Your FACS operator should be able to set the correct gating strategy to exclude dead/damaged cells. if you have difficulties with this you can contact me directly and I''ll be happy to advise.

Normally there is no need to use viability discriminator for fresh PBMCs unless the blood has been kept around for more than few hours before processing or has been stimulated; harsh RBC lysis might require the use of viability dye too. However should you wish to look for antigens expressed at very low frequency (less than 1%) or intracellularly the use of a discriminator is advisable, to better separate background reactivity from true staining. For use with intracellular staining you will need to use a "fixable" viability dye, since the fixation process will allow Propidium or similar to penetrate the cell membranes.

good luck, Mara

Thanks very much for your very helpful answers! The reason I am interested in long term storage is that I wont have the funds for the flow cytometry until after I've collected the samples (could be a delay of 1-3 years). This is part of a study I am setting up and I haven't actually collected the samples as yet. Are there any tips for how best to store samples for eventual use in FC? I am worried the cells will burst if frozen and then defrosted after several years. Again thanks for your advice.

The best solution would be to find working flow cytometer somewhere near from you and to establish collaboration. It would be preferable to analyze samples immediately.

Given that setting up a good antibody panel for immunophenotyping can also be tricky (depending on what and how many parameters you want to look at), I would absolutely recommend to involve somebody who has some experience with multicolour FACS analysis and preferably a machine that you can use.

I have no experience with freezing whole blood, but PBMCs can be easily frozen in e.g. FCS with 10% DMSO. If you freeze them down carefully, cell death should not present a major problem (again, this is not the case for granulocytes). Some companies (e.g. Nalgene) offer specialised freezing containers which ensure that cells are cooled down at optimal rates and this can help to improve sample quality.

U can go for double strength FCS+10%DMSO. handle the cells very gently once its isolated. You can use PBMCs preferably, specially for subsets immuno phenotyoing using cell surface markers.

You please take good care tht once you add DMSO in to the cells, you should immediately transfer the cells in to frosty or isopropanol bath and keep it in -80degree C. let it be in -80 for two to three days and then shift it to liquid nitrogen container. keeping cells in -80 prior to shifting to -121 degree C ie., liquid nitrogen will keep cells safe from bursting as snap cooling is not done here.

it can be stored for 4-7yrs..i can assure 95% viability provided the cells should be isolated within 2-3hrs from sample collection, gentle handling and proper storage and thawing in 37 degree C. Water bath set at 37degree C would be appropriate.

hope this helps. In case you need any help reg protocol, feel free to contact.

all the best.. Greshma :)