Fitc conjugated anti mouse cd80

Isolated pulmonary macrophages were incubated with FITC‐conjugated anti‐mouse CD80 (BD Biosciences, CA, USA), APC‐conjugated anti‐mouse F4/80 (BD Biosciences), and PerCP‐Cy5.5‐conjugated CD206 (BD Biosciences). Analysis was performed by ImageStreamx Mark II flow cytometer (Merck, Darmstadt, Germany). M1 macrophages were classified as F4/80+/CD80+ and M2 macrophages were identified as F4/80+/CD206.

Phycoerythrin (PE)-conjugated anti-mouse CD19 (Clone: 1D3), FITC-conjugated anti-mouse MHC class II (I-A/I-E), FITC-conjugated anti-mouse CD80 (Clone: 16-10A1), Hamster IgG2 κ isotype control FITC, FITC-conjugated anti-mouse CD86 (Clone: GL1), Rat IgG2a κ isotype control FITC antibodies were purchased from BD Biosciences. Purified anti-mouse TLR1 (Clone: eBioTR23), Rat IgG2a κ isotype control purified, Anti-rat IgG Biotin, Streptavidin PE, FITC-conjugated anti-mouse TLR2 (Clone: 6C2), Rat IgG2b κ isotype control FITC, PE-conjugated anti-mouse TLR4 (Clone: UT41), Mouse IgG1 κ isotype control PE antibodies were obtained from eBioscience. Purified anti-mouse TLR6 (Clone: 418601) was purchased from R&D Systems. Detoxi-Gel Endotoxin Removing Columns were purchased from Pierce. Antibodies to phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, phospho-IκB, Lamin B1 and α-Tubulin were obtained from Cell Signaling Technology. α-p65, IκB and β-Actin antibodies were purchased from Santa Cruz Biotechnology. Biotin-Conjugated Anti-Phosphotyrosine antibody (clone 4G10) was obtained from Millipore.

RAW264.7 or macrophages from WT or TLR2^−/− mouse were incubated with PPE26 (10μg/ml), Pam₃CSK4 (5 mg/ml), or LPS (1μg/ml) for 36 h. Then, the cells were harvested and washed with prechilled PBS, followed by centrifugation at 1000 x g for 10 min at 4°C. The cells were treated with Fc Block (1:100) (BD Pharmingen, CA, USA) in PBS supplemented with 1% BSA and incubated with PE-conjugated anti-mouse CD86, FITC-conjugated anti-mouse CD80, PE-conjugated anti-mouse H-2κB for mouse macrophages, or FITC-conjugated anti-mouse I-A/I-E (BD Pharmingen, CA, USA) on ice for 1 h in the dark room. The cells were resuspended in 500μl PBS and analyzed using a flow cytometer (Becton Dickinson, USA). The data were analyzed using the Cell-Quest data analysis software (10000 events per sample) and Flow4J.