F4 80 clone bm8

PECs or blood leukocytes were blocked in Fcγ block (1 μg/ml; eBioscience) and then surface stained with fluorochrome-conjugated antibodies against Ly6G (clone 1A8; BD Bioscience), F4/80 (clone BM8; eBioscience), CD11c (clone N418; eBioscience), and CD11b (clone M1/70; eBioscience). Flow cytometric data were acquired with a BD FACSCanto II instrument (BD Biosciences) and analyzed using FlowJo software (Tree Star).
To assess the rate of S. aureus phagocytosis by BMDCs, cells that had been infected with CTV-labeled S. aureus for 30 min or 2 h were incubated with gentamicin (200 μg/ml) for 1 h, washed, and fixed in 2% PFA. The cells were then analyzed on BD FACSCanto II by gating on forward scatter and side scatter, and the percentage of CTV⁺ cells was assessed.

Rac2 antibody is from Novus Biologicals. Rac1 antibody is obtained from Santa Cruz Biotechnology. Vitronectin and collagen are from Sigma (Sigma-Aldrich, St. Louis, MO), fragments of fibronectin (H296 and CH271) are from R&D systems. Primary or fluorescent antibodies against CD31 (clone MEC13.3), CD11b (clone M1/70) are from BD Biosciences, F4/80 (clone BM8) is from eBiosciences. 4, 6 diamidino-2-phenylindole (DAPI) are obtained from Sigma. Alexa Flour 594 or Alexa Flour 488 is from Invitrogen life Technologies. Collagenase/Dispase is from Roche Biosciences, hyaluronidase type V and Dnase I is from Sigma. α-isonitrosopropiophenone for arginase activity and sulphanilamide for nitrite assay is from Sigma. Bouin's solution is from Sigma. MCSF is from Gibco Life technologies and GMCSF from Peprotech Life sciences. D-luciferin potassium salt is from Caliper Life sciences. CD11b magnetic beads are from Militenyi Biotec.RNA isolation kit from Qiagen. Iscript cDNA synthesis kit and SYBR green are from Biorad (Bio-Rad, Hercules, CA).

The following antibodies were used for flow cytometry: CD11b-PeCy7 (clone M1/70, eBioscience), F4/80 (clone BM8, eBioscience), and fixable viability dye (eBioscience). The following antibodies were used for western blotting: phospho-p65, NF-κB2, phospho-ERK, phospho-JNK, phospho-p38, total ERK, total JNK, and total p38 from Cell Signaling Technologies. Total p65 was purchased from Santa Cruz. TRAF2, gasdermin D and caspase-11 were purchased from Abcam, caspase-1 from Adipogen and IL-1β from RnD. cIAP1 was purchased from Human Atlas. RIPK1 and XIAP was purchased from BD Biosciences. Secondary antibodies for western blotting such as donkey anti mouse/rabbit/rat IgG conjugated to HRP are from SouthernBiotec, the donkey anti goat IgG was purchased from Santa Cruz. The neutralizing antibody against TNF (at 200 ng/mL, MP6-XT22) was purchased from BioLegend.

Liver tissue samples were fixed in 10% formaldehyde for 24 hours and stored in PBS. The fixed tissues were processed routinely, embedded in paraffin, cut into 5 μm thickness and stained with hematoxylin and eosin (H&E). For immunohistochemistry (IHC), the tissue sections were subjected to deparaffination, 3% H₂O₂ treatment and antigen retrieval, and then primary antibody F4/80 (clone BM8, eBioscience, San Diego, CA) and HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (KPL, Gaithersburg, MD). Staining was developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and nuclei were stained by hematoxylin. Three non-overlapping areas were obtained and averaged per slide (x 200 magnification) with a microscope (Olympus IX-51).

Renal tissue was fixed in 10% formaldehyde, embedded in paraffin, and mounted onto slides for staining. Hematoxylin/Eosin stains were used to evaluate renal tubular injury scores. Briefly, random fields were semiquantitatively evaluated separately by two blinded investigators (×200 magnification) with the following scoring system: 0 = no injury; 1 = 1–25% of area injured; 2 = 25–50%; 3 = 51–75%; and 4 = 76–100%. Immunolabeled antibodies for F4/80 (clone BM8, eBioscience), Ly6G (clone 1A8, Biolegend), complement component C3/C3b (Abcam), alpha smooth muscle actin (clone EPR5368, Abcam-Epitomics) and Desmin (Abcam-Epitomics). For collagen staining slides were deparaffinized and immersed in picrosirius red (Sigma-Aldrich) solution for 1 hour, dehydrated in 100% ETOH and cleared in xylene. Images were analyzed blindly by two separate investigators and quantification was performed using ImageJ software (NIH, Bethesda, MD). Positive-staining area and cell counts for each image were quantified using color-separation and background-subtraction, automatic thresholding and particle-analysis algorithms as previously published [26 (link)].

BALF samples from mice were obtained as described above. After centrifugation at 1,600 rpm for 5 min at 4°C, cell pellets were seeded in a 24-well plate (Corning, USA) and incubated for 12 h at 37°C 5% CO₂ in DMEM medium containing 10% fetal bovine serum (FBS). Nonadherent cells were removed by rinsing the wells with PBS, and the adhered cells were detached by adding 0.25% Trypsin/EDTA (Gibco) after washing three times with 1× PBS. To determine the purity of the adhered cells (AMΦs), cells were stained with antibodies directed to F4/80 (clone: BM8, eBioscience, USA) and CD11b (clone: M1/70, Biolegend, USA) and analyzed by flow cytometry. The purity of AMΦs was ≥ 95%.