摘要
目的比较实验室配制的红细胞裂解液的裂解时间对流式测定SD大鼠和食蟹猴外周血T淋巴细胞亚群的影响。方法(1)取15只食蟹猴的肝素钠抗凝血各50μl,血样与荧光检测抗体Per CP-CD3/FITC-CD8/PE-CD4孵育后分别经自制和市售红细胞裂解液裂解一段时间,比较样本经自制与市售裂解液处理不同时间后其CD3~+CD4~+,CD3~+CD8~+和CD3~+淋巴细胞的平均荧光强度(median fluorescence intensity,MFI)等数值的差异;(2)取15只SD大鼠的EDTA-K2抗凝血各50μl,血样与荧光检测抗体APC-CD3/FITC-CD4/PE-CD8a孵育后,分别经自制红细胞裂解液和市售裂解液裂解一段时间,比较样本经自制与市售裂解液处理不同时间后CD3~+CD4~+和CD3~+CD8~+淋巴细胞的MFI等数值的差异;(3)取15只大鼠EDTA-K2抗凝血各50μl,血样与荧光检测抗体APC-CD3/PE-CD4孵育后,分别经自制红细胞裂解液和市售裂解液裂解一段时间,比较样本经自制与市售裂解液处理不同时间后PE-CD4~+的MFI差异。结果(1)食蟹猴外周血经自制红细胞裂解液,裂解时间约15min和5min的FITC-CD8~+的MFI分别为1745±173和2439±253,淋巴细胞百分比分别为47.9%±7.2%和42.1%±7.6%;裂解时间约15min和5min的PE-CD4~+的MFI分别为12924±892和13695±984,淋巴细胞百分比分别为38.7%±7.2%和33.1%±7.2%。裂解时间约15min比裂解5min的FITC-CD8~+(t=8.779,P<0.0001)和PE-CD4~+(t=2.250,P<0.05)的MFI降低;淋巴细胞百分比占比(t=2.139,2.162,均P<0.05)增高,差异具有统计学意义。食蟹猴外周血经市售红细胞裂解液裂解15min,FITC-CD8~+,PE-CD4~+,Per CP-CD3~+的MFI分别为2413±240,12428±1208和1015±123。与自制裂解液裂解5min比较,市售裂解液裂解15min的PE-CD4~+和Per CP-CD3~+的MFI降低,差异具有统计学意义(t=3.150,4.862,均P<0.01)。(2)SD大鼠外周血经自制红细胞裂解液,裂解时间5min和2min的FITC-CD4~+的MFI分别为55.72±14.10和225.08±12.44;裂解5min与裂解2min的FITC-CD4~+的MFI比较差异有统计学意
Objective To compare the effect of the lysis time of lab-prepared erythrocyte lysate onflow cytometric determination of peripheral blood T lymphocyte subsets in SD rats and cynomolgus monkeys.Methods①About 50μl of heparin sodium anticoagulated blood from 15 cynomolgus monkeys was taken,the blood sample was incubated with thefluorescent antibodies PerCP-CD3/FITC-CD8/PE-CD4 and lysed by erythrocyte lysate for a period of time,the effect of lysis time on the meanfluorescence intensities(MIF)of CD3+CD4+and CD3+CD8+lymphocytes determined byflow cytometry was compared.②About 50μl of EDTA-K2 anticoagulant blood from 15 SD rats,the blood sample was incubated with thefluorescent antibodies APC-CD3/FITC-CD4/PE-CD8a and lysed by erythrocyte lysate for a period of time,the effect of lysis time on the MIF of CD3+CD4+and CD3+CD8+lymphocytes determined byflow cytometry was compared.③About 50μl of EDTA-K2 anticoagulant blood from 15 SD rats,the blood sample was incubated with thefluorescent antibodies APC-CD3/PE-CD4 and lysed by erythrocyte+lysate for a period of time,the effects of lysis time on the MFI of PE-CD4 lymphocytes determined byflow cytometry was compared.Results①The peripheral blood of cynomolgus monkeys was lysed by self-made erythrocyte lysate and the MFIs of+FITC-CD8 with lysis time of about 15 min and 5 min were 1745±173 and 2439±253 respectively,and the percentage of+lymphocytes was 47.9%±7.2%and 42.1%±7.6%.The MFIs of PE-CD4 with lysis time of about 15 min and 5 min were 12924±892 and 13695±984,and the percentage of lymphocytes was 38.7%±7.2%and 33.1%±7.2%.The MFI of in FITC-++CD8(t=8.779,P<0.0001),PE-CD4(t=2.250,P<0.05)and percentage of lymphocytes(t=2.139,2.162;all P<0.05)lysed for 15min were significantly lower than those lysed for 5min,and the differences were statistically significant.There was also a++difference in the percentage of FITC-CD8 and PE-CD lymphocytes(t=2.131,P<0.05).The peripheral blood of cynomolgus monkeys was lysed by commercially available erythrocyte lysate,a
作者
姜华
黄瑛
王超
秦超
孙立
韩素芹
王三龙
文海若
JIANG Hua;HUANG Ying;WANG Chao;QIN Chao;SUN Li;HAN Suqin;WANG Sanlong;WEN Hairuo(Beijing Key Laboratory,National Center for Safety Evaluation of Drugs,National Institutes for Food and Drug Control,Beijing 100176,China;School of Phamaceutical Sciences,Sun Yat-sen University,Guangzhou 510006,China)
出处
《现代检验医学杂志》
CAS
2023年第5期165-170,共6页
Journal of Modern Laboratory Medicine
基金
国家重点研发计划课题(2021YFA1101602):干细胞及相关治疗产品质量控制和非临床评价关键技术与规范研究
中国食品药品检定研究院关键技术研究基金(GJJS-2022-6-1):脐带间充质干细胞和CD19嵌合抗原受体T细胞非临床有效性评价方法及药效学机制研究。
