Bs 0293g af488

For the detection of surface expression of MHC-II or the location of NF-κB, cells were incubated with primary antibodies of CD86 (212927, Elabscience, China), CD206 (B354282, Biolegend, United States), NF-κB (10745-1-AP, Proteintech, Wuhan, China), and MHC-II (sc-66205, SANTA CRUZ BIOTECHNOLOGY, Santa Cruz, America) overnight at 4 C, and then incubated with goat anti-rat IgG/Alexa fluor 488 secondary antibodies (bs-0293G-AF488, Bioss, Beijing, China) for another 120 min. After incubating with fluorescent secondary antibodies, the cells were washed three times before flow cytometry or LSCM.

For the detection of NF-κB, LC-3, and MHC-II expressions or location, cells were incubated with primary antibodies of NF-κB (10745-1-AP, Proteintech, Wuhan, China), LC-3 (12741S, CST, Boston, United States), CD80 (E-AB-F0992D, Elabscience), CD86 (E-AB-F0994D, Elabscience), and MHC-II (sc-66205, Santa Cruz Biotechnology, Santa Cruz, United States) overnight at 4°C and then incubated with the goat anti-rat IgG/Alexa Fluor 488 secondary antibody (bs-0293G-AF488, Bioss, Beijing, China) for another 60 min and washed three times before confocal microscopy (FV3000RS, Olympus, Japan) or flow cytometry (CytoFLEX, Beckman Coulter, United States). CD80 and CD86 were direct-labeled antibodies, and no secondary antibody incubation was required.