Abstract:

Objective To investigate the effect of heat denaturation on labeled peripheral blood cell subsets and establish a stable protocol of flow cytometry-Fluorescence in situ hybridization (Flow-FISH). Methods Heparinized fresh peripheral blood samples were taken from ten healthy persons. T lymphocytes, B lymphocytes and monocytes were labeled with Alexa Fluor^®647-CD3, CD19 and CD14 seperately, then denatured by high temperature. The expression of lineage-specific antigens were detected by flow cytometry, on cells with and without heat denaturation separately. T lymphocytes were labeled with Alexa Fluor^®647-CD3, and were allowed to react with four concentrations of crosslinker Bis[sulfosuccinimidyl] suberate (BS³) seperately, and the efficiency was evaluated by flow cytometry. Results After heat denaturation, the expression of CD3 and CD19 became weaker, while T lymphocytes and B lymphocytes could still be differentiated effectively; the expression of CD14 did not change, and monocytes could be differentiated clearly. After the reaction with BS³ in four concentrations, the percentages of Alexa Fluor^®647-CD3 labeled T lymphocytes were similar. The application of Alexa Fluor^®647 labeled anti-CD3, CD19 and CD14 antibodies might make the differentiation of blood cell subsets by Flow-FISH feasible, and the optimal final concentration of BS³ would be 2.5 mmol/L.

Key words: heat denaturation, cell subset, crosslinker, flow cytometry