Percp cy5.5 conjugated anti mhc 2

C57BL6 mice were purchased from National Institute of Nutrition and maintained in a pathogen-free condition at NIAB animal facility. The animal care and all the experimental procedures were performed in accordance with the guidelines approved by Institutional Animal Ethics Committee (IAEC) of National Institute of Animal Biotechnology (NIAB). A mouse macrophage cell line (RAW264.7) human embryonic kidney cell line (HEK293) were originally purchased from the American Type Culture Collection (Manassas, VA). WT (NR- 9456), TLR2^−/−(NR-9457), TLR4^−/−(NR-9458), TLR2^−/−/4^−/− DKO (NR-19975) cell lines were obtained from BEI Resources, USA. Cells were cultured in DMEM (Sigma, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), penicillin (100 U/ml), and streptomycin (100 mg/ml) and maintained at 37 °C in a humidified incubator (5% CO₂). Anti-TLR2 and anti-TLR4 were purchased from Biolegend, USA and the inhibitors NF-kB (SN50), p38 (SB203580) and JNK (SP600125) were from Invivogen and Sigma-Aldrich (St. Louis, MO). Human and mouse IL-8, IL-6 and TNF-α sandwich ELISA kits were from R&D biosystems. PerCP-CY5.5–conjugated anti MHC-II, PE-conjugated CD80, APC conjugated CD86 and BV421- CD40 for flow cytometry experiments were procured from BD biosciences, US. All reagents were purchased from Sigma unless otherwise specified.

RAW264.7 (10⁶ cells per well) were stimulated with LPS (500 ng/ml) or PMB-treated rLenB, LenD, rLenE, rLsa30, rLipl21, or rLoa22 for 24 h at 37°C in presence of 5% CO₂. Cells were harvested and washed with prechilled PBS and after blocking; they were then incubated with PerCP-CY5.5–conjugated anti-MHCII, APC-conjugated CD80, and PE-conjugated CD86 (BD Biosciences, United States) for 1 h on ice in the dark. The cells were fixed with 1% paraformaldehyde, and 50,000 total events per sample were acquired using a LSRFortessa. The data were analyzed using FlowJo software.