Proil 1β clone njten3

Cells were isolated as described above from inguinal, abdominal and thoracic mammary glands. Following isolation, cells were resuspended in full medium (RPMI (Seraglob), 10% FBS (Gibco), 0.01 M HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 2 mM Glutamax (Gibco), 1% Penicillin–Streptomycin (Gibco), 1% nonessential amino acids (Gibco), 57.2 μM β-mercaptoethanol (AppliChem)) with GolgiPlug and GolgiStop 1:1,000 (BD) and exposed to Zymosan 50 µg ml^–1 (InvivoGen) or LPS 300 ng ml^–1 (Sigma) or left unstimulated (negative control). The cells were incubated for 6 h at 37°C, washed and stained for surface markers as described above, then fixed with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C, washed with Perm buffer (2% BSA, 0.5% Saponin, 0.0002% sodium azide in PBS), stained with intracellular antibody mix in Perm buffer for 25 min (TNF (clone MP6-XT22 1:400) and proIL-1β (clone NJTEN3 1:200), purchased either from eBioscience or Biolegend), washed with Perm buffer, resuspended in PBS and acquired.

Single cell suspensions were washed and resuspended in FB. For intracellular cytokine analysis, cells were resuspended in 500 μL of eBioscience^TM Cell Stimulation Cocktail (plus protein transport inhibitors) in RPMI medium supplemented with 10% FCS, 1% l‐glutamine, and 0.01% β‐mercaptoethanol and the technique was carried out as previously outlined [53 (link)]. These cells remained at 37°C, 5% CO₂ for 4 h before being washed in FB, centrifuged at 400 × g for 5 min at 4°C and subjected to surface staining as above. Following overnight incubation in Fix/Perm buffer, cells were washed twice with 2 mL Perm Buffer (both Foxp3/Transcription Factor staining buffer set, eBioscience). Cells were then resuspended in 200 μL of Perm Buffer supplemented with intracellular antibodies (1:200 for each) for 1 h in the dark. Antibodies used were against: Ki67 (clone 16A8, Biolegend), IFN‐γ (clone 554413, BD Biosciences), IL‐6 (clone MP520F3, BD Biosciences), TNF‐α (clone MP6‐XT22, Biolegend), IL‐12p23 (clone C15.6, Biolegend), pro‐IL‐1β (clone NJTEN‐3, eBioscience). Appropriate isotype controls for intracellular stains were included in all experiments.

The following antibodies were used for flow cytometry analysis: CD11b (clone M1/70) was purchased from BD Biosciences; MHCII (clone M5/114.15.2), Tim4 (clone RMT4‐54), F4/80 (clone BM8), Ly6G (clone 1A8) from BioLegend; pro‐IL‐1β (clone NJTEN3) and matching isotype control antibody were purchased from eBioscience. For extracellular staining, cells were collected and resuspended in flow cytometry buffer (4% FCS in PBS) containing 4 μg/ml of Fc receptor blocking antibody 2.4G2 (homemade) for 15 min on ice. Cells were then stained for 30 min with the indicated antibodies. For intracellular staining, cells were first fixed for 15 min with 2% paraformaldehyde (PFA) and then permeabilized and blocked at 4°C for 30 min in permeabilization buffer (0.5% bovine serum albumin (BSA), 5 mM EDTA, 2 mM NaN₃ and 0.5% saponin) containing 4 μg/ml 2.4G2 blocking antibody (homemade). Cells were then stained with the indicated antibodies for 1 h at 4°C in permeabilization buffer. Flow cytometry was performed on Cyan (Beckman Coulter) or Attune (Thermo Fisher) flow cytometer and analysed with FlowJo software.