Fcr blocking reagent

After euthanasia, the skin of irradiated mice was shredded using a sharp blade. Subsequently, the skin was incubated in DMEM containing 0.5 mg/mL collagenase I (A004194, Diamond), 0.5 mg/mL collagenase II (A004174, Diamond), 1 mg/mL collagenase IV (A004186, Diamond), 0.5 mg/mL hyaluronidase (H3506, Biosharp), 0.5 mg/mL elastase (A002290, Diamond) and 12.5 kU DNase (D4263, Sigma). Incubation was performed at 37 °C for 90 min. After incubation, cells were filtered through a 70 μm cell strainer. Subsequently, cells were treated with FcR blocking reagent (156604, Biolegend) for 10 min at 4 °C, then stained with fluorescently labeled CD26 antibody, and dead cells were filtered using DAPI. Senescent cellular fibroblasts with DAPI⁻ CD26⁺ tdTomato⁺ and non-senescent fibroblasts with DAPI⁻ CD26⁺ tdTomato⁻ were isolated using a BD FACS Aria II, and culture was continued in DMEM.

To generate a single-cell suspension from the skin, subcutaneous fat was removed, and the skin was chopped into 1-mm pieces which were then incubated in liberase (300 µg/mL; Cat# 5,401,119,001, Sigma-Aldrich, Darmstadt, Germany) and DNase 1 digestion cocktail (50 U/mL, Cat# Roche-11284932001, Sigma-Aldrich, Darmstadt, Germany) for 60–90 min at 37 °C as described [28 (link)]. Digested skin cells were passed through a 100-µm pore filter and then through a 40-µm pore filter. The single cells obtained were washed once in 1% BSA and then blocked with FcR blocking reagent (Cat# 101,302, BioLegend, San Diego, USA) for 10 min. Thereafter, cells were incubated with following antibodies; anti-P-cadherin (Cat# AF761, R&D Systems, Minnesota, USA), anti-CD49f-APC (Cat# 17–0495-82, Invitrogen, Frankfurt, Germany), anti-CD45-Vio-Blue (Cat# 130–092-910, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD-90.2-PE (Cat# 130–102-489, Miltenyi, Bergisch Gladbach, Germany), and CD31 (Cat#DIA 310, clone: SZ31, Dianova, Hamburg, Germany). An Aria III cell sorter (BD Biosciences, Heidelberg, Germany) was used to sort the hair matrix cells (PCAD + CD49f + CD45-CD90.2- CD31-CD326-).

BMDMs were treated with FcR Blocking Reagent (BioLegend), and incubated with PE F4/80 (BioLegend, 111703), PE/Cy7 CD11b (BioLegend, 101215), and FITC CD86 (BioLegend, 105005) antibodies. Next, cells were fixed with Fixation Buffer (BioLegend) and permeated with Intracellular Staining Permeabilization Wash Buffer (BioLegend). APC CD206 (BioLegend, 141707) antibody was then used to incubate cells for flow cytometry assay. Macrophages were firstly screened based on the expression of F4/80 and CD11b, and CD86 (M1 marker) and CD206 (M2 marker) expressions were then determined. Data were analyzed by Flowjo Analysis Software.

Single-cell suspensions were generated from tumor extracts through physical (gentleMACS Octo Dissociator, Miltenyi) and enzymatic (Liberase DL, Millipore Sigma LIBDL-RO) dissociation. Cell concentrations were determined and 1 × 10⁶ cells per sample measured were subjected to viability (LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, L34957), CD45-FITC (11-0451-82), CD4-Brilliant Violet™ 421 (404-0042-82) and CD8-PE (12-0081-82; all reagents from Thermo Fisher Scientific) staining in the presence of FcR blocking reagent (Biolegend 101320) in V-bottom, 96-well plates. The frequency of CD4⁺ and CD8⁺ T-cells was determined in the singlet, live, CD45⁺ population by analytical flow cytometry analysis on a MACS Quant Analyzer 10 instrument (Miltenyi).

Tumor nodules were treated with Tumor & Tissue Dissociation Reagent (Becton Dickinson, Franklin Lake, NJ, USA). The resulting cell suspensions were treated with FcR-blocking reagent (BioLegend), then the cells were reacted with APC anti-mouse PD-L1 antibody (10F.9G2), PE anti-mouse PD-L2 antibody (TY25), PE/Cyanine7 anti-mouse F4/80 antibody (BM8), Violet 51 anti-mouse/human CD11b antibody (M1/70), or isotype-matched control IgGs (all from BioLegend). Dead cells were excluded by labeling with Fixable Viability Dye eFluor™ 780 (Invitrogen). The stained cell samples were analyzed on a FACSverse (Becton Dickinson) flow cytometer with FlowJo software (Becton Dickinson).

To analyze expression of TREM1 in monocytes and TAMs, cells were pre-incubated with FcR blocking reagent (Biolegend, USA, 422302) 15 min at 4°C. Then cells were stained with anti-human CD354 (TRME-1)-PE (Biolegend, USA, 314906) antibodies for 30 min at 4°C in the dark. Cells were washed with PBS twice, and suspended in PBS for detection. The samples were acquired on a FACSAria flow cytometer (BD Biosciences) and the data were analyzed with FlowJo software.

We used flow cytometry to evaluate the phenotypic expression of PBMCs from NPC patients and healthy donors. In general, PBMCs were blocked with an FcR‐blocking reagent (Biolegend) to avoid nonspecific binding, and then stained with corresponding antibodies for 30 min at 4°C. For FOXP3 staining, cells were fixed and permeabilized with FOXP3 fix/perm buffer set from eBioscience (ThermoFisher Scientific) according to the manufacturer's protocols. Fixable Viability Dye eFluor 506 was used to distinguish between dead and living cells. Cells were run through a CYTOFLEX flow cytometer (Beckman Coulter) and results were analyzed using Flow Jo software. Antibodies used are detailed in Table S2.