Fitc anti human cd69 antibody

Lamina propria mononuclear cells (LPMCs) were isolated from intestinal resection samples as previously described.⁶⁴ (link) The dead cell removal kit (#130-090-101, Miltenyi Biotec) was used to remove dead cells from LPMCs. The magnetic-activated cell sorting and fluorescence-activated cell sorting were used to obtain pure cell populations. CD4⁺ T_RM cells were isolated from the LPMCs by negative selection using human CD4⁺ T-cell memory effector cell isolation kit²³ (link) (#130-094-125, Miltenyi Biotec). In brief, the magnetically labeled B cells, naive T cells, antigen-presenting cells, CD8⁺ T cells, non-αβ⁺ T cells, and effector and central memory CD4⁺ T cells were positively selected. Then, CD4⁺ T_RM cells in LPMCs were purified using fluorescence-activated cell sorter according to established surface markers CD69 and CD103 (FITC anti-human CD69 antibody, BioLegend 310904; PE/cyanine7 anti-human CD103 antibody, BioLegend 350212).²² (link) For the ex vivo culture of purified CD4⁺ T_RM cells, complete X-VIVO 15 hematopoietic cell medium was obtained from Lonza (#BE02-060Q).