He Zhengxin, Chen Jing, Wang Xianling, Zhao Bohua, Hou Tianwen
Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang 050082, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 Apr;31(4):470-3.
To establish a whole blood leukocyte phagocytosis assay for Candida albicans (C.albicans) based on flow cytometry (FCM).
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.
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.
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.
建立基于流式细胞术（FCM）的全血白细胞对白色念珠菌（白念珠菌）的吞噬试验。
用荧光探针羧基荧光素二乙酸琥珀酰亚胺酯（CFDA-SE）标记对数生长期中期的白念珠菌，然后以10:1的感染复数（MOI）加入预先用CD45-PC5染色的人全血细胞中，于37℃孵育。细胞分别孵育10、30和60分钟。通过流式细胞术测定血液中CD45阳性细胞对白念珠菌的吞噬率。
在酵母提取物蛋白胨葡萄糖培养基（YPD）中，于37℃和5% CO2条件下，白念珠菌SC5314的对数生长期为第5至11小时。经30分钟孵育后，10 mmol/L CFDA-SE能很好地对白念珠菌进行染色。SC5314白念珠菌与CD45⁺细胞孵育10、30和60分钟后，流式细胞术测得中性粒细胞的吞噬率分别为（80.1±6.1）%、（83.8±7.7）%和（92.3±11.2）%，单核细胞的吞噬率分别为（11.2±3.6）%、（15.8±4.4）%和（27.7±6.8）%，淋巴细胞的吞噬率分别为（0.9±0.3）%、（0.8±0.4）%和（5.2±1.6）%。
成功建立了基于流式细胞术的全血白细胞对白念珠菌吞噬作用的检测方法，30分钟是吞噬试验的适宜孵育时间。
