Whole blood leukocyte phagocytosis assay for Candida albicans based on flow cytometry

OBJECTIVE: To establish a whole blood leukocyte phagocytosis assay for Candida albicans (C.albicans) based on flow cytometry (FCM). METHODS: C.albicans of mid-logarithmic growth phase was labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), and then added into CD45-PC5 pre-stained human whole blood cells at a 10:1 multiplicity of infection (MOI) in 37DegreesCelsius. The cells were incubated for 10, 30 and 60 minutes. Phagocytosis rate of C.albicans by the CD45 positive cells in the blood was determined by FCM. RESULTS: In yeast extract peptone dextrose medium (YPD) and under the conditions of 37DegreesCelsius and 50 mL/L CO2, the logarithmic growth phase of C.albicans SC5314 was from the 5th to 11th hour. C.albicans were well stained by 10 mmol/L CFDA-SE after 30-minute incubation. After 10-, 30- and 60-minute incubation with SC5314 C.albicans with CD45⁺ cells, the phagocytosis rates measured by FCM were (80.1 ± 6.1)%, (83.8 ± 7.7)% and (92.3 ± 11.2)% for the neutrophils, (11.2 ± 3.6)%, (15.8 ± 4.4)% and (27.7 ± 6.8)% for the monocytes and (0.9 ± 0.3)%, (0.8 ± 0.4)% and (5.2 ± 1.6)% for the lymphocytes. CONCLUSION: The method for measuring whole blood leukocyte phagocytosis of C.albicans based on FCM is successfully established, and 30 minutes are the proper incubation time for the phagocytosis assay.