Pe conjugated anti hmgb1 antibody

Flow cytometry analysis was used to measure phospho-mTOR (p-mTOR; activated mTOR), p21 (senescence-associated marker), S100A8/9, and HMGB1 (SASP-associated markers) expression levels in lung cells. Single cells prepared from lung tissue were analyzed by intracellular staining with FITC-conjugated anti-S100A8/9 antibody (Novus Biologicals, CO), PE/Cy7-conjugated anti-p-mTOR antibody (Thermo Fisher Scientific, MA), and APC-conjugated anti-p21 antibody (BD Biosciences, Santa Cruz, CA). For flow cytometry analysis, at least 1 × 10⁵ cells were acquired using an LSR II (BD Biosciences). All data were analyzed using FlowJo ver. 10 software (BD Biosciences). To measure S100A8/9 and HMGB1 expression levels in various lung cells, single cells were first stained with PerCPCy5.5-conjugated anti-CD45 antibody, BV711-conjugated anti-CD4 antibody, BV506-conjugated anti-F4/80 antibody, APC/Cy7-conjugated anti-CD11c antibody, and BV421-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody, then with FITC-conjugated anti-S100A8/9 antibody and PE-conjugated anti-HMGB1 antibody (Thermo Fisher Scientific) for intracellular staining.

Single cells prepared from lung tissue were stimulated with phorbol 12-myristate 13-acetate (100 ng/mL), ionomycin (1 μg/mL), and Golgi stop, then stained with PerCP-Cy5.5-conjugated anti-cluster of differentiation (CD) 45 antibody (Thermo Fisher Scientific). To quantify Th1, Th2, or Th17 cells, cells were stained with BV711-conjugated anti-CD4 antibody and PE-conjugated anti-IFN-γ, PE-Cy7-conjugated anti-IL-13, APC-conjugated anti-IL-5, or BV421-conjugated anti-IL-17A antibodies. To measure S100A8/9 and HMGB1 expression in various lung cells, single cells were stained with PerCP-Cy5.5-conjugated anti-CD45 antibody, BV711-conjugated anti-CD4 antibody, BV506-conjugated anti-F4/80 antibody, APC/Cy7-conjugated anti-CD11c antibody, and BV421-conjugated antiepithelial cell adhesion molecule (EpCAM) antibody, followed by intracellular staining with FITC-conjugated anti-S100A8/9 antibody and PE-conjugated anti-HMGB1 antibody (Thermo Fisher Scientific).