[Analysis of the storage time in paraformaldehyde-fixed T cells and their subsets in Kunming mice]

Objective To determine the expiration dates of CD3e⁺CD4⁺ T and CD3e⁺CD8a⁺ T lymphocytes of Kunming mice. Methods The sterile spleen and anticoagulant blood of Kunming mice were collected and used to prepare the single-cell suspensions. After CD3e, CD4 and CD8a of T cells were stained with fluorophore-conjugated monoclonal antibodies, the samples were fixed with paraformaldehyde (PFA) and stored at 4DegreesCelsius in dark followed by detection using three-color flow cytometry at several specific time points. The best regression curves fitted with the least squares method were used for calculating the contents of T cells at each time point and the subsequent statistical analysis. Samples without PFA served as the blank controls. Results The levels of PFA-fixed CD3e⁺CD4⁺ T and CD3e⁺CD8a⁺ T lymphocytes from spleen tissues significantly decreased at 11 days and 16 days post collection, respectively; whereas no difference was found in the samples without PFA until 8 days and 11 days post collection, respectively. As for the T cells separated from the peripheral blood, the longer storage periods were both 18 days under the PFA fixation, which were longer than those of the blank controls (10 and 15 days, respectively). Conclusion The PFA-fixed T lymphocytes of the spleen and peripheral blood of Kunming mice should be detected using flow cytometry within 11 days post collection.