Fitc anti mouse h 2kb clone af6 88.5

L929 cells (ATCC) and HeLa cells from American Type Culture Collection stably transfected with a cDNA encoding the MHC class I K^b molecule were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7.5% fetal bovine serum (FBS) at 37 °C with 9% CO₂. 293T and MDCK cells from American Type Culture Collection were propagated in DMEM supplemented with 10% FBS at 37 °C with 9% CO₂.
Spliceostatin A was synthesized as described (14 (link)). Cycloheximide (Sigma), anti-β-actin antibody (Sigma), MG132 (Calbiochem) were obtained from vendors. Anti-SIINFEKL C-terminal antibody (1F10.2.2) was obtained from Kenneth Rock (U Mass Med). Anti-IAV mAbs were described previously (15 (link)): anti-HA, H36-26; anti-NA, NA2-1C1; anti-M1, M2-1C6; anti-M2, O19 (16 (link)). The anti-NA rabbit polyclonal Ab used for western blotting was described (17 (link)). For flow cytometry, FITC anti-mouse H-2K^b (clone#AF6-88.5) was from BD Biosciences. MAb 25D1.16 (anti-K^b-SIINFEKL) and M2-1C6 were labeled with Alexa Fluor 647 (Life Technologies). NA2-1C1 was labeled with Pacific Blue (Life Technologies). H36-26 and O19 were labeled with Alexa Fluor 488 (Life Technologies).

BMDCs were infected with Salmonella at multiplicities of infection (MOI) of 0,1.25, 2.5, 5, and 10. After incubation for 0, 0.5, 1, or 2 h, which allow the cells to phagocytize the bacteria, gentamicin (final concentration 20 μg/mL) was added to the culture to stop extracellular replication of Salmonella in the culture media. Then BMDC/Salmonella cultures were incubated for an additional 14 h. Cell viability (%) and expression of CD11c, MHC I, and MHC II in the BMDC cultures were measured by flow cytometry using fixable viability stain 510 (BD Biosciences), PE-anti mouse CD11c (clone HL3, BD Biosciences), FITC-anti mouse H-2Kb (clone AF6–88.5, BD Biosciences), and V450-anti mouse I-A/I-E (clone M5/ 114.15.2, eBioscience).