Cd11b percp cy5

HDM was from Greer Laboratories. Anti-mouse IL-1β (p17) (AF-401-NA) was from R&D. Anti-mouse caspase-1 (p20) (AG-20B-0042) and anti-NLRP3 (AG-20B-0014) antibodies were from Adipogen. Anti-ASC (67824) antibody was from Cell Signaling Technology. Anti-β-actin (66009-1-Ig) was from Proteintech.
Anti-mouse antibodies used for flow cytometry were: CD3-FITC (BD, 553062, 145-2C11), CD19-FITC (eBioscience, 11-0193-82, 1D3), Ly-6G-PE (Biolegend, 127608, 1A8), CD45-PE (eBioscience, 12-0451-81, 30-F11), CD11c-PerCP-Cy5.5 (Biolegend, 117328, N418), CD11b-PerCP-Cy5.5 (BD, 550993, M1/70), CD11b-PE-Cy7 (eBioscience, 25-0112-82, M1/70), CD3e-PE-Cy7 (BD, 552774, 145-2C11), CD19-PE-Cy7 (Biolegend, 115520, 6D5), SiglecF-Alexa Fluor 647 (BD, 562680, E50-2440), MHCII-APC-eFluor 780 (eBioscience, 47-5321-82, M5/114.15.2), Ly-6G-BV421 (Biolegend, 127628, 1A8), CD45-BV510 (Biolegend, 103137, 30-F11), CD11c-BV510 (Biolegend, 117338, N418). Ultrapure LPS was obtained from Invitrogen. Nigericin was obtained from Sigma-Aldrich. RRx-001 (S8405) was from Selleck.

Anti-mouse antibodies to CD45-FITC, CD11b-PerCP-Cy5.5, CD11c-APC, Ly6G-BV421, Siglec-F-PE, and fixable viability stain 780 were obtained from BD Pharmingen. The lungs from mice were perfused and digested into single-cell suspensions as described previously (57 (link)). After RBC lysis buffer treatment, the whole lung cells were washed with PBS and then stained with corresponding fluorescent antibodies. Following incubation, samples were washed and fixed in 2% ultrapure formaldehyde. The absolute number of each leukocyte subset was then determined by multiplying the absolute number of CD45⁺ cells by the percentage of cells stained by fluorochrome-labeled antibodies for each cell population analyzed using BD FACSArray software™ on a BD FACSArray flow cytometer (BD Biosciences, San Jose, CA, USA). AMs were identified as CD45⁺CD11c⁺Siglec-F⁺. Neutrophils were identified as CD45⁺CD11b⁺Ly6G⁺.
Cell sorting was performed on BD FACSAria II instrument, using BD FACSDiva software (BD Biosciences), and compensation and data analyses were performed using FlowJo software (TreeStar, Ashland, OR, USA). Cell populations were identified using sequential gating strategy. The sorting purity was 90–95%.

Splenocytes were isolated under aseptic conditions from all mice groups and layered on Ficoll Hypaque for density gradient separation of the splenic lymphocytes. These splenic lymphocytes were stained for various cell surface and intracellular markers such as CD3, CD4, CD8, CD19, CD11b, F4/80, and RANKL. Briefly, Aliquots of 1 × 10⁶ splenic lymphocytes were washed with ice-cold 1XPBS and fixed with 1% paraformaldehyde for 15 min at 4°C. The cells were washed with FACS buffer (1XPBS + 2% FBS + 0.001% sodium azide) and then labeled with fluorophore tagged antibodies CD3-PB, CD4-PECF594, CD8-PE, CD19-FITC, CD11b-Percp cy5.5 (BD Biosciences, USA), and F4/80 APC-cy7, RANKL-PE (BioLegend, San Diego, USA). Further, the cells were washed with FACS buffer and acquired on FACS Aria (BD Biosciences San Jose, USA). Data analysis was done using FlowJo software (Tree Star, Ashland, USA).

After isopycnic centrifugation with Percoll, a portion of the collected cell suspensions (~5 × 10⁵ cells) was stained with combinations of fluorochrome-conjugated antibodies against CD11b, CD68, and F4/80: F4/80-PE (Biolegend), CD11b-PerCP-Cy5.5 (BD Biosciences), and CD68-Alexa Fluor 684 (AbD Serotec). Fluorochrome-labeled isotype identical antibodies served as control. After 25 min of incubation at 4°C, cells were washed and percentage of Kupffer cell subtypes and amount of NF-κB activation (EGFP, FITC channel) were determined using FACSCalibur flow cytometer (BD Biosciences).

Intestinal lamina propria cells and MLN cells were labeled with CD4-FITC, CD25-APC (eBioscience), CD8-APC-Cy7, CD3-PerCP (Biolegend), and B220-BV570 (BD Biosciences). Labeling of intracellular FoxP3 was performed after extracellular staining and fixation/permeabilization of cells. GM-CSF-derived dendritic cells were labeled with CD11b-PerCP Cy5.5 (BD Biosciences), CD11c-PE-Cy7, I-A/I-E-APC-Cy7, CD80-PE, CD86-APC, CD8-FITC, and B220-BV570 (Biolegend). Flt3L-derived dendritic cells were labeled with CD11c-PE-Cy7, CD11b- or Siglec-H-PerCP Cy5.5 (Biolegend), I-A/I-E-APC-Cy7, CD317-PE, CD40-Alexa Fluor 647, CD103-FITC, and B220-BV570. Coculture T cells were labeled with CD4-PerCP Cy5.5 (BD Biosciences), CD127-PE-Cy7, CD73-APC, CD195 (CCR5)-FITC, CD62L-BV570, and CD25-APC-Cy7 (Biolegend).

BALF and serum were collected as described previously [9 (link)]. Differential cell counts were calculated, and total BALF-protein was measured by using the Pierce BCA Protein Assay Kit (Thermo Scientific, USA) according to the provider’s manual. BALF protein and cell differentials (total cells, neutrophils and macrophages) from mice with 24 h LPS exposure were presented previously in Kling et al. [9 (link)]. BALF macrophages were further subject to characterization by flow cytometry. BALF macrophage samples of two animals from the same exposure group were pooled in the 24 h exposure experiment (resulting in n = 3-5). However, at the 72 h time point all BALF samples were tested individually. BALF cells were first blocked with anti-CD16/32 (BioLegend) and 5% fetal calf serum (FCS) and then stained for 30 min on ice with antibodies from BioLegend (CD11b-PerCP/Cy5.5, CD11c-FITC, CD11c-APC, CD80-PE, CD86-APC, CD86-FITC) and BD Biosciences (SiglecF-APC/Cy7) and measured with a FACS Canto II flow cytometer (BD Biosciences). Alveolar macrophages were selected as CD11c⁺, SiglecF⁺ and CD11b^low cells and expression of CD80 and CD86 was assessed as mean fluorescent intensity (MFI).