The question is qhy are you keeping them at 4 C, 37 C, or room temp. Depending on the processes, you may or may not see internalization or changes in CD expression.

I have a feeling that Keeping a Long time on Room Temp or 4 C may cause apoptosis and necrosis and may effect your experiments. Best way may be stain lymphocytes with antibody and fix it with fixative (4% formalin/paraformal dedyde/alcohol, depend upon cell and experiments). You can stored these fixed lymphocyte at 4 C for flow cytometry analysis later on (upto 3-4 day or a week). If you want to do sorting then i recommend you to use immediately.

The answer is: it depends on the CDs you are interested in. Some molecules are very stable and will remain on the cell surface for days. Others will be internalized and expression will be lost as quickly as a few hours after draw.

Internalization occurs faster at 37 than at RT than at 4C. Blood (contrsry to popular belief and practice) can be stored at 4C, however the longer it is stored at 4C the higher the cell loss will be as will CD expression

They are going to either proliferate or die at room temperature. You can fix them with 1% paraformaldehyde for flow cytometry and refrigerate.

The answer as always is it depends. For just looking at surface staining and fixing (1% PFA is fine 1% formalin in 1% BSA (FACS FIX)works too) is the best. These can be stored at 4 for a long time- as much as a week. There are some changes in the FSC vs SSV following fixation, but if you controls are treated correctly there is no problem. You cannot use PI as a live/dead discriminator on fixed cells. Some antibodies will work on prefixed cells others will not.

For functional analysis, (IFN-g etc) you almost are better off freezing the cells for 4 days. We have done a bunch of CTL analysis and 2 days after draw is about as long as the cells will reproducibly respond. We found leaving the cells in EDTA tubes at RT is the best, but certainly not perfect and not beyond 48 hours. 4 has a lot of cell death.

Are they sorted/isolated lymphocytes from whole blood by Ficol? I would definitely put in the culture with 10% human A/B serum/RPMI for 3 days (without any extra cytokines, i.e., IL-2) and perfrom flow analysis unless there is some specific reason that you can not fix the cells as suggested by others.

IF lympocytes of interest are from human whole blood, one can also stored THE WHOLE blood in vacutainer/collection bag at 37oC temperature with a gentle circular movement until isolation of the lympocyte for the analysis 3-4 days later.

And I agree with Jeffrey that 48 hrs are about the max for the RT storage of whole blood.

Are been the lymphocytes isolated from whole blood by Ficol?

Here it was better make flow analysis to define surface phenotype at TIME ZERO.

After. Are been the lymphocytes plated in continuous culture at 37 °C in an incubator with 5% CO2?

I would certainly put the lymphocytes divided in two culture flasks at 5 X 105 cells per ml. All tested cells can be suspended in RPMI 1640 with L-glutamine (or in D-MEM High Glucose) with 10% human serum (collected in dry tubes from the same donor of lymphocytes, for not adding variables) and cultured for 4 days, without change the culture media.

It is very important that the cell concentration remains stable.

In first condition I would definitely put culture medium without any extra cytokines.

In second condition I would definitely added IL-2 at 25 ug/L.

These conditions may allow to evaluate the phenotypic change of lymphocytes in continuous culture with or without extra-cytokines.

Max storage time of whole blood at RT should be 24-48 hrs (preferably not more than 24 hours at RT). Depending on the incubation times, surface CD can change for possible internalization or catabolism.

For this reason it is better make flow analysis by FACS (fixed cells) or by Sorting (in vivo cells) to define the surface phenotype at TIME ZERO, as soon as the lymphocytes were isolated from whole blood.

For functional analysis, cells are better if maintained into microenvironment of incubator (at 37°C and 5% of CO2, to avoid an increase in the rate of cell death or cell dysfunction that increase at room temperaturethat increase at room temperature without CO2 adding) but certainly not beyond 48 hours. On the other hand, it is preferable that the supernatants are collected and kept at 4 ° C.

Please, see:

Cell Biochem Funct. 1999 Mar;17(1):47-55.

Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type-1.

Thank you four your helps friends