A method for detection of monocyte/macrophage phagocytosis of fluorescent labeled Mycobacterium tuberculosis by flow cytometry

Objective: To establish a method for detection of monocyte/macrophage phagocytosis of fluorescent labeled Mycobacterium tuberculosis(Mtb) by flow cytometry.Methods: Mtb was labeled with fluorescein isothiocyanate(FITC) at different concentrations.The efficient labeled rate was detected by flow cytometry.Whole blood taken from healthy subjects were incubated with FITC labeled Mtb at 37℃ from 10 min to 120 min,followed by lysing red blood cells,washings,and then quenched the fluorescein of Mtb that not phagocytosed with trypan blue.The amount of monocytes with positive FITC were measured as phagocytosis rate by flow cytometry.FITC labeled Mtb and phorbol myristrate-differentiated THP-1 cells as macrophages were incubated from 1 h to 6 h with the ratio of 10:1,or incubated for 1 h and 2 h at the ratios from 1:1 to 100:1,and the phagocytosis rates were detected by flow cytometry as same method above.Results: The efficient labeled rate for Mtb was 92% when incubated with FITC at the concentration of 50 μg/ml.The percentages of monocyte phagocytosis of Mtb were increased from 34.68% to 79.90% in coculture time from 10 min to 120 min.The percentages of THP-1 macrophage phagocytosis of Mtb were increased from 30% to 81% for 1 h and 6 h of coculture.The THP-1 cell phagocytosis of Mtb were reached platform when FITC labeled Mtb incubated with THP-1 cells with at the ratio of 100:1 for 1 h(81%),or at the ratio of 50:1 for 2 h(82%).After quenched with trypan blue,the percentages of monocyte phagocytosis of Mtb decreased by 29% in 10 min and by 6% in 120 min,whereas the percentages of THP-1 cell phagocytosis of Mtb decreased by 1% in 1 h and 7% in 6 h.Conclusions: The application of flow cytometry to measure monocyte/macrophage phagocytosis of FITC labeled Mtb is simple,rapid,and reproducible method.