Fitc annexin 5 apoptosis detection kit with propidium iodide

Apoptosis of BMDM were determined by using FITC Annexin V apoptosis detection kit with Propidium Iodide (PI) (BD biosciences), MHC-II expression of BMDM in response to rRv1768 stimulation was measured by PerCP/Cy5.5 anti-MHC-II (Biolegend, CA, USA). Cytokines production in culture supernatant of rRv1768 stimulated BMDM was determined using mouse Th1/Th2/Th17 cytometric-beads array kit (BD PharMingen, USA), according to the manufacturer's protocol. Intracellular IFN-γ expression of CD3⁺CD4⁺ T cells was determined by staining with APC-anti-CD3, FITC-anti-CD4, PE-anti-IFN-γ (Biolegend, CA, USA). All procedures were performed as we previously described (Tang et al., 2017 (link); Yuan et al., 2019 (link)) and analyzed on BD Accuri C6 Flow cytometer (BD Biosciences).

Cardiomyocyte apoptosis was evaluated using the FITC Annexin V Apoptosis Detection kit with propidium iodide (PI) (BD Pharmingen, San Diego, CA, USA). The cardiomyocytes were cultured in 20 μL binding buffer; PI (5 μL) and annexin V-FITC (10 μL) were added to the cardiomyocyte suspension. The cells were then incubated at room temperature in the dark for 30 min. Flow cytometric analysis was performed using a FACScan device (Beckman Coulter, Fullerton, CA, USA). The data were analyzed using FlowJo software (BD Biosciences, San Jose, CA, USA).