Vaccine and Infectious Disease Organization, 120 Veterinary Road, University of Saskatchewan, Saskatoon, SK, S7N 5E3, Canada.
Vaccine and Infectious Disease Organization, 120 Veterinary Road, University of Saskatchewan, Saskatoon, SK, S7N 5E3, Canada; School of Public Health, University of Saskatchewan, Saskatoon, SK, S7N 2Z4, Canada.
Vet Immunol Immunopathol. 2021 Dec;242:110352. doi: 10.1016/j.vetimm.2021.110352. Epub 2021 Nov 9.
An effective method to isolate functional eosinophils from blood and tissues is required to analyze the multiple roles eosinophils play in innate immunity and tissue homeostasis. Highspeed cell sorting was used to isolate bovine eosinophils from blood polymorphonuclear (PMN) cells and from small intestine intraepithelial leukocytes. Eosinophils and neutrophils were purified from bovine blood with highspeed cell sorting after gating on autofluorescence (FL1) high and low PMN subpopulations. Highspeed sorting of intestinal eosinophils was accomplished by using a combination of positive (CD45, CD11c, side scatter) and negative (CD3) selection parameters. Eosinophils sorted from blood PMNs were 88.6 ± 5.8 % (mean + 1 SD; n = 4) pure and yielded significantly (p < 0.05) more RNA than purified neutrophils. Analysis of Toll-like receptor (TLR) gene expression and TLR ligand-induced pro-inflammatory cytokine (IL-1, IL-6, IL-8, and TNFα) gene expression demonstrated significant (p < 0.01) functional differences between blood eosinophils and neutrophils. Eosinophils varied between 14.7 % to 29.3 % of CD45 IELs and purity of sorted intestinal eosinophils was 95 + 3.5 % (mean + 1SD; n = 5). A comparison of mucosal and blood eosinophils revealed significant (p < 0.01) differences in TLR gene expression, supporting the hypothesis that functionally distinct eosinophil populations are present in blood and tissues. In conclusion, highspeed cell sorting provides an effective method to isolate viable eosinophils from blood and tissues that can then be used for transcriptome analyses and in vitro function assays.
从血液和组织中分离功能正常的嗜酸性粒细胞是分析嗜酸性粒细胞在先天免疫和组织稳态中发挥的多种作用所必需的。高速细胞分选用于从血液多形核（PMN）细胞和小肠上皮内白细胞中分离牛嗜酸性粒细胞。通过对自动荧光（FL1）高和低PMN 亚群进行门控，使用高速细胞分选从牛血液中纯化嗜酸性粒细胞和中性粒细胞。通过使用正（CD45、CD11c、侧向散射）和负（CD3）选择参数的组合，完成肠嗜酸性粒细胞的高速分选。从血液 PMN 中分选的嗜酸性粒细胞纯度为 88.6±5.8%（平均值+1SD；n=4），比纯化的中性粒细胞产生的 RNA 显著（p<0.05）多。分析 Toll 样受体（TLR）基因表达和 TLR 配体诱导的促炎细胞因子（IL-1、IL-6、IL-8 和 TNFα）基因表达表明，血液嗜酸性粒细胞和中性粒细胞之间存在显著（p<0.01）的功能差异。嗜酸性粒细胞在 CD45 IEL 中的比例为 14.7%至 29.3%，分选的肠嗜酸性粒细胞的纯度为 95+3.5%（平均值+1SD；n=5）。比较黏膜和血液嗜酸性粒细胞表明 TLR 基因表达存在显著差异（p<0.01），支持了在血液和组织中存在功能不同的嗜酸性粒细胞群体的假说。总之，高速细胞分选为从血液和组织中分离活的嗜酸性粒细胞提供了一种有效方法，然后可用于转录组分析和体外功能测定。
