Anti human cd45 ko j33

Spleen and mesenteric lymph nodes (LN) were harvested and cells were obtained by mashing over a 40 µm filter. Single cell suspensions of spleen and LN were phenotypically analyzed using a Navios multi-color flow cytometer (Beckman-Coulter, Mijdrecht, the Netherlands). Human leukocytes were identified using an antibody directed against the common human surface leukocyte marker CD45 (anti-human-CD45-KO (J33, Beckman-Coulter). For cell-surface staining of human CD4 and CD8 T cells, the following antibodies were used: anti-CD4-PC5.5 (13B8.2, Beckman Coulter), and anti-CD8-APC-AF700 (B9.11, Beckman-Coulter). For intracellular staining with anti-FOXP3-e450 (PCH101, eBioscience), cells were fixed and permeabilized by fix-perm treatment (eBioscience) according to manufacturers instruction. Data were analyzed using Kaluza software version 1.5a (Beckman-Coulter) and gates were set based on single staining and FMOs (Supplemental Fig. 1)

Muti-color flow cytometry of peripheral blood mononuclear cells (PBMCs) was performed in the Department of Clinical Laboratory, Sun Yat-sen Memorial Hospital, Sun Yat-sen University (Guangzhou, China). Cells were stained for surface markers with the following fluorochrome-conjugated monoclonal antibodies (mAbs): antihuman CD69-PE (FN50), antihuman CD16-PE (B73.1), antihuman CD25-PE-Cy7 (2A3), antihuman CD56-PE-Cy7 (NCAM16.2), antihuman HLA-DR-APC (L243), antihuman CD19-APC-H7 (SJ25C1), antihuman CD8-APC-H7 (SK1), and antihuman CD38-V450 (HB7), which all were purchased from BD Biosciences (Franklin Lakes, NJ, USA); antihuman CD3-FITC (UCHT1), antihuman CD127-PE (R34.34), antihuman CD4-PC5.5 (13B8.2), and antihuman CD45-KO (J33) antibodies were obtained from Beckman Coulter Inc. (Brea, CA, USA). All stained samples were acquired with a 10-color flow cytometer (Navios, Beckman Coulter Inc.), and data were analyzed by Kaluza 2.1.1. software (Beckman Coulter Inc.). The gating strategies are summarized in Supplementary Figure S1.