Apc cd45 antibody

The cell suspension of was collected as mentioned above. The cell pellet was dyed with FVS570 (564995, BD Pharmingen) and resuspended in FACS, added with CD16/32 (553142, BD Pharmingen), and incubated at 4 °C for 15 minutes. Then we performed flow cytometry staining using BV421-CD279 antibody (135217, Biolegend) and APC-CD45 antibody (559864, BD Pharmingen). We analyzed the cells using the Beckman CytoFlex S machine.

SVF cells were isolated from the iWAT of 4-week-old Lats1^fl/fl; Lats2^fl/fl; Rosa-LSL-tdTomato (Con) and Adipoq-Cre; Lats1^fl/fl; Lats2^fl/fl; Rosa-LSL-tdTomato (AKO) mice. The SVF pooled from 3–5 mice per genotype was sorted using a flow cytometer (BD Aria II) to exclude CD45⁺ leucocytes as previously described^{21 (link)}. Briefly, the SVF underwent RBC lysis (11814389001, Roche) for 3 min, before washing with HBSS/3% BSA. Subsequently, cells were incubated with APC-CD45 antibody (559864, BD Pharmingen) at a 1:200 ratio diluted in HBSS/3% BSA for 20 min on ice, washed with HBSS/3% BSA and resuspended in FACS buffer (PBS/0.5% BSA). CD45⁻/tdT⁺ cells from the AKO SVF and CD45⁻/tdT⁻ cells from the control SVF were sorted for single-cell RNA-seq. In total, 5,000 single-sorted cells were targeted and processed for single-cell library generation using 10x Chromium Single Cell 3′ reagent kit v3 (10x Genomics), following the manufacturer’s protocols. Single-cell libraries were sequenced on the HiSeq-X platform (Illumina).

As we have described before, BAL was performed by instilling 5 mL of serum free DMEM media. BAL fluid was incubated in 24-well plates for 2 hours at 37 °C and 5% CO₂. The attached cells were analysed by flow cytometry, and more than 80% of the cells were positive for CD11b (BD Biosciences, San Jose, CA, USA) and F4/80 (BD Biosciences, San Jose, CA, USA). Isolated alveolar macrophages were stimulated with bleomycin (25 μg/ml) and treated with Chol-HCQ (10 μM) or HCQ (10 μM) and etanercept (5 μg/ml) in the presence of brefeldin A (GolgiPlug, BD Biosciences, San Jose, CA, USA) in a 24-well plate at 37 °C for 24 h. For macrophage intracellular cytokine detection, cells were stained with APC-CD45 antibody (BD Biosciences, 1:100), PerCP-Cy5.5-CD11b antibody (BD Biosciences, San Jose, CA, USA, 1:100) and FITC-F4/80 antibody (BD Biosciences, San Jose, CA, USA, 1:100) for 30 min in PBS at 4 °C and then washed twice with PBS. The cells were fixed in a 2% paraformaldehyde solution for 20 min, permeabilised using 1% Tritox-100 for 30 min at 4 °C and washed with PBS. Then, the cells were stained with PE-TNF-α antibody (BD Biosciences, San Jose, CA, USA, 1:100) for 2 h at 4 °C, washed, and examined with a FACS Caliber Flow Cytometer (BD Biosciences, San Jose, CA, USA).