Fitc conjugated anti mouse tlr2

RAW 264.7 cells were harvested in ice cold buffer (PBS with 2% newborn calf serum band 0.05% sodium azide) and labelled with rat monoclonal anti-mouse MHC class II non-polymorphic region (NIMR-4) FITC (Abcam), FITC-conjugated anti-mouse TLR2 (eBioscience) or respective isotype controls and fixed in 1% paraformaldehyde, as previously described [44 (link)]. Data were acquired on a FACSCalibur flow cytometer (BD Biosciences). Expression of MHC-II on the positively stained population (cells staining for the respective antibodies) was analysed using CellQuest Pro software (BD Biosciences). Data are presented as the median fluorescence intensity (MFI) for the specific antibody minus the MFI for the isotype control antibody.

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.