Pe anti mouse inos clone cxnft

Cells were cultivated in a 3.5 cm glass bottom confocal dish (SPL Life Sciences, Pocheon, Korea) at a density of 1.2 × 10⁶ cells per dish. After incubating for 24 h, the cells were fixed with 4% paraformaldehyde for 20 min and blocked with 10% bovine serum albumin (BSA) for 20 min. The cells were incubated with conjugated antibodies eFluor 660 anti-mouse F4/80 (, clone BM8, eBioscience) (1:100), PE anti-mouse iNOS (clone CXNFT, eBioscience) (1:300), and DAPI (4′,6-diamidino-2-phenylindole, Invitrogen, Carlsbad, CA, United States) for 1 h. Images were captured using a laser scanning confocal microscope (LSM 880, Carl Zeiss, United States) with a ×40 objective (original magnification).

Cells were scraped and stained with the following antibodies: APC-eFluor 780 anti-mouse F4/80 (clone BM8, eBioscience, San Diego, CA, United States), Alexa Fluor 488 anti-mouse CD11b (clone M1/70, BD Biosciences), PE anti-mouse iNOS (clone CXNFT, eBioscience), and eFluor450 anti-mouse CD38 (clone 90, eBioscience). The isotype controls were rat IgG2bκ (eBR2a) and rat IgG2bκ (DA/HA) from BD Biosciences (San Jose, CA, United States). The cells were washed and stained with Flow Cytometry Staining Buffer (eBioscience) for 30 min at 4°C. For intracellular staining, cells were fixed in IC Fixation Buffer (eBioscience) for 30 min at 4°C, permeabilized, and stained in permeabilization buffer (eBioscience). Results were obtained using the FACSVerse (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, United States). The F4/80⁺ and CD11b⁺ cells were gated as fully differentiated macrophages.