Fluorochrome conjugated anti cd11b

For analyzing subset proportions, the monocytes treated with LPS for 5 days were harvested and incubated with anti-CD16/-CD32 antibodies (1:100 dilution, BD Biosciences, no. 553141) to block Fc-receptors. The cells were stained with fluorochrome-conjugated anti-CD11b (1:200 dilution, BioLegend, no. 101226) and anti-Ly6C (1:200 dilution, BioLegend, no. 128006) antibodies, and PI (Sigma-Aldrich) was added before flow cytometry. For examining S100A8 expression, the monocytes were treated with high-dose LPS (1 µg/mL), and IRF7 siRNA (Thermo Fisher Scientific), IRF3 siRNA (Thermo Fisher Scientific), or control siRNA (Thermo Fisher Scientific) was mixed with Lipofectamine reagent to transfect monocyte cultures (25 pmol siRNA/well). Fresh LPS and siRNA were added every two days. After 5 days, the cells were fixed, permeabilized, stained with fluorochrome-conjugated anti-S100Ab antibody (1:100 dilution, Novus, no. NBP2-27067AF647), and then analyzed by flow cytometry.

Fluorochrome–conjugated anti–CD11b and anti–F4/80 were purchased from Biolegend. Cell death was identified using a violet Live/Dead Kit or 7AAD staining (Invitrogen). The cells were stained with fluorescence conjugated antibodies in 2% (vol/vol) FBS in PBS at 4°C for 30 min. Data were collected using a BD FACSAria II and analyzed using the FlowJo software. CD11b^intF4/80⁺ KCs from C57BL/6J mice were sorted using a BD FACSAria II sorter.