Isotype control igg

When the percentage of CPCs that had adhered to the slide was 90%, cells were prepared into a single-cell solution. Then, they were stained with either an anti-c-kit (eBioscience, San Diego, CA) or IgG isotype control (eBioscience, San Diego, CA) PE-conjugated antibody. The positive rate of c-kit or isotype control in the total cell population was detected by FACS Calibur flow cytometry.

The expression of αvβ₃, αvβ₅, and α₅β₁ integrin dimers on cell surface was analyzed by fluorescence-activated cell sorting (FACS) analysis on live cells. Cells were removed from the dish with TrypLE (GIBCO, Grand Island, NY, USA), centrifuged in cold media, and the pellet was washed with cold flow cytometry (FC) buffer (PBS containing 0.1% sodium azide and 2 mM EDTA). Cells were incubated with phycoerythrin-conjugated primary antibodies for αvβ₃ (EMD Millipore, Temecula, CA, USA), αvβ₅ (R&D, Minneapolis, MN, USA), and the IgG-isotype control (eBioscience, San Diego, CA, USA). Unconjugated antibody was used for staining α₅β₁ integrin (EMD Millipore). All primary antibodies were incubated for 1 hr at 4°C. Alexa Fluor 594-conjugated secondary antibody for 30 min at 4°C was used for α₅β₁ integrin. After antibody incubations, cells were washed three times with FC buffer, and live cells were analyzed on the BD FACSCalibur using the Cell Quest software.

BMDCs or MoDCs were harvested and resuspended in complete RPMI 1640. A total of 2 × 10⁵ cells per well were seeded into 48-well tissue culture plates. C. difficile filtrate was diluted 1:20 into the BMDC suspension, and the remaining volume was made up with complete culture medium (containing toxins A and B where indicated). Purified toxins A and B were a kind gift from Techlab, Inc. (Blacksburg, VA). Recombinant serum amyloid A (PeproTech), TLR ligand-tested lipopolysaccharide (Sigma), anti-mouse IL-1β monoclonal antibody (clone B122; eBioscience), IgG isotype control (eBioscience), recombinant IL-1 receptor antagonist (R&D Systems), recombinant murine IL-1β (R&D Systems), YVAD-FMK (Enzo Life Sciences), and glybenclamide (InvivoGen) were diluted in RPMI 1640 and added to cells as indicated. For incubation of BMDCs with excess potassium, 0.8 M stock solutions of KCl and NaCl were prepared, filter sterilized, and diluted 1:20 into the BMDC suspension. Stimulated cells were incubated for 24 h at 37°C with 5% CO₂. Cells were spun at 300 × g for 5 min, and the supernatant was harvested and frozen at − 80°C. Remaining cells were washed in 1× PBS once and lysed in an appropriate volume of RLT buffer (lysis buffer of the RNeasy kit; Qiagen). RNA-containing lysates were frozen at −80°C until RNA isolation.