Pe conjugated anti tlr2

Renal tubular epithelial cells were grown in collagen plates as indicated above. Cells were trypsinized and counted. 1×10(x002C6)6 were added to FACS tubes and spun down at 1200 RPM for 5 minutes. Media was removed and cells resuspended in 400μL of FACS wash buffer. Cells were then divided evenly into individual wells of a Costar round bottom 96-well plate (Corning, Corning, NY) and stained with TruStain FcX (Biolegend; San Diego, CA) in FACS wash buffer at 1:50 dilution and placed on ice for 5 minute incubation. 50μL of PE-conjugated anti-TLR2 (eBiosciences; San Diego, CA) in FACS wash buffer (1:50 dilution) was then added to cell suspensions (final dilution of 1:100) and placed on ice away from light for 45 minutes incubation. Stained cells were washed two times with FACS wash buffer and placed in 1.2mL micro titer tubes on ice (Fischer Scientific; Hampton, NH) for FACS analysis. Cells were run on the BD Biosciences LSRIII and analysis performed on FlowJo software.

PerCP-conjugated anti-CD115, FITC-conjugated anti-CD45, Fixable Viability dye eFluor 780, PerCP-conjugated anti-CD64, PE-conjugated anti-TLR2, PE-conjugated anti-TLR4/MD2, PE-conjugated anti-CD29 and all matching isotypes were purchased from eBioscience (San Diego, CA). Alexa647-conjugated anti-Siglec-F, PE-conjugated anti-CD11b, FITC-conjugated anti-GR-1, APC-conjugated anti-streptavidin Ab, PE-conjugated anti-CD18 (clone C71/16) and Fc Block were obtained from BD Biosciences (Bedford, MA). Biotinylated anti-CD31 Ab was obtained from Biolegend (San Diego, CA). Rat anti-mouse anti-gp96 Ab was purchased from Enzo Life Sciences (Farmingdale, NY) and anti-rat IgG HRP from Santa Cruz (Dallas, TX). Biotinylated anti-F4/80 was obtained from Bio-Connect (Huissen, The Netherlands). CD18 blocking ab (purified NA/LE rat anti-mouse CD18; clone: GAME-46) and matching control Ab for in vivo studies were purchased from BD Biosciences.