Multitest 4 color tbnk reagent

The immune cells for the flow cytometry analysis were collected at the time of admission and transported to our testing center within 2 hours. PBMCs were then isolated and analyzed. Peripheral lymphocyte subpopulations were detected with BD Multitest 4-color TBNK reagent. Flow cytometric phenotyping of expression of selected activation markers (CD38 on CD8⁺ cells, HLA-DR on CD3⁺ cells, CD38 and HLA-DR co-expression on CD8⁺ cells) in 33 collected samples were detected with a BD FACS Caliber Flow Cytometer (BD Biosciences, San Diego, CA, USA) as previously described (15 (link)). The gating strategies are illustrated in Supplementary Figures 1-2. The number of lymphocytes used in each flow cytometry analysis was ensured to be above 2500.

In the retrospective part, the percentages and absolute counts of lymphocytes as well as CD4⁺ and CD8⁺ subpopulations of T cells in peripheral blood were tested with BD Multitest 4-color TBNK reagent (Catalog No. 340503), and a BD FACS Caliber Flow Cytometer (BD Biosciences, San Diego, CA, USA) was used for analysis. To evaluate the levels of RIPK1 and RIPK3 in peripheral blood lymphocytes of AOSD patients, blood samples were obtained from 24 AOSD patients and 20 healthy controls (HCs). First, 100 µl peripheral blood was lysed by erythrocyte lysate; the remaining cells were then washed with PBS and fixed with BD fixation/permeabilization solution (Catalog No. 554714) for 20 min. After fixation/permeabilization, cells were washed by BD perm/wash buffer. Then, intracellular RIPK1/3 was stained with rabbit IgG anti-human RIPK1/3 antibody (Abcam, catalog No. 178420 for RIPK1 and US Biological, catalog No. 041063 for RIPK3) or control rabbit IgG and incubated for 30 min. Cells were then washed twice with PBS, and stained with a secondary phycoerythrin (PE)-conjugated anti-rabbit IgG antibody (BD Bioscience) for 30 min avoid from light. After washing, cells were suspended with 500 μl PBS and then analyzed on a BD FACS Aria II Flow Cytometer; the data were analyzed using FlowJo software (TreeStar, Inc., San Carlos, CA, USA).