Pe mouse igg1 κ isotype control

Parental and OxR cell lines were cultured to 70% confluency upon collection and split into 250,000 cells per sample. Cells were fixed in 4% PFA in HBSS for 15 min at RT, then blocked in a 100 μl 1% BSA solution for 30 min at 4°C, with 2× HBSS washes between each step. Cells suspensions of 100 μl were incubated for 15 min at RT with 2 μl Human TruStain FcX (BioLegend, 422301) to prevent nonspecific Fc receptor binding. Samples were immediately stained with 5 μl of either PE anti-human CD261 (DR4) (BioLegend, clone DJR1), PE anti-human CD262 (DR5) (BioLegend, clone DJR2-4), PE anti-human TRAILR3 (DcR1) (BioLegend, clone DJR3), PE anti-human TRAILR4 (DcR2) (R&D Systems, clone 104918), or PE Mouse IgG1 κ Isotype Control (BioLegend, clone MOPC-21) for 30 min at 4°C. Samples were washed twice with HBSS and analyzed using a Guava easyCyte flow cytometer. A chi-squared test was performed using FlowJo v10.7.1, where significance in histogram distribution was confirmed if T(x) between parental and OxR stained samples was greater than T(x) between background (unstained) parental and OxR samples (see Supplementary file 1).

TGF-β1 (PEPRO TECH, China, 100-21-10 μg), PC-MSC (Provided by Shenzhen 150 Biomedical Co.,Ltd.), ESC (Provided by ATCC), FITC Mouse IgG1 κ Isotype control (Biolegend, 7313762), PE Mouse IgG1 κ Isotype control (Biolegend, 9217868), APC Mouse IgG1 κ Isotype control (Biolegend, 9282592), PE Mouse Anti-Human CD73 (Biolegend, 8151901), APC Mouse Anti-Human CD105 (Biolegend, 9277133), FITC Mouse IgG2a κ Isotype control (Biolegend, 8241935), FITC Mouse Anti-Human CD14 (Biolegend, 8299630), FITC Mouse Anti-Human CD45 (Biolegend, 8206704), DMEM/F12 medium (Peiyuan, Shanghai, L310 kJ), Fetal bovine serum (Gemini, 900-108), Trypsin digestive fluid (Beyo, C0203), DPBS (Beyo, C0221G), CCK8 (Beyotime, C0039), EDU staining kit (Beyotime, C0081s), Hematoxylin-eosin staining kit (Solarbio, G1120), DEPC-treated water (Solarbio, R1600), trichloromethane (Sinopharm Chemical Reagent Co. LTD., 10006818), GelRed dye (Biotium, #41003), RNA Loading Buffer (TaKaRa, 9168), Tris-Borate-EDTA Buffer (TBE) 10× Powder, pH8.3 (TaKaRa, T9122), RNAsimple Total RNA Kit (TIANGEN, DP419), All-in-One First-Strand Synthesis Master Mix (Yugong Biolabs, EG15133S), SuperReal PreMix Plus (SYBR Green) (TIANGEN, FP205-02), and Methacrylate anhydride (MAA, 0.1 mL/1 g gelatin), ELISA Kit (Solarbio, SEKH-0052), MateRegen^® Gel (2104006, BioRegen Biomedical (Changzhou) Co., Ltd.).

FITC mouse IgG1 κ isotype control (400110), PerCP-Cy5.5 mouse IgG1 κ isotype control (400149), PE mouse IgG1 κ isotype control (400114), PerCP-Cy5.5 anti-human CD3 (300430), FITC anti-human CD8a (300906), PE anti-human CD45 (368510), PerCP-Cy5.5 anti-human IFN-γ (506528), FITC Rat IgG2a κ isotype control (400506), PerCP-Cy5.5 Rat IgG1 κ isotype control (400426), FITC anti-mouse CD3 (100204), FITC anti-mouse CD8a (100705), PE anti-mouse CD45 (103106), and PerCP-Cy5.5 anti-mouse IFN-γ (505822) were purchased from Biolegend. PE anti-human PD-L1 (557924), FITC anti-human PD-L1 (558065), FITC anti-human HLA-A2 (343303), FITC Rat IgG2b κ isotype control (400605), PE Rat IgG2a λ isotype control (400635), and PE anti-mouse PD-L1 (558091) were obtained from BD Biosciences.

After different neutrophil subgroups were obtained from step 2.2.1.2, typically 1 × 10⁵ cell/50 μl and the following anti-human fluorochrome-conjugated mAbs or specific isotype controls: CD66b (Biolegend, USA), PE-Cy7 mouse anti-human CD10 (Biolegend, USA), PerCP-vio700 mouse anti-human CD11b (Miltenyi, German), APC mouse anti-human CD16 (Biolegend, USA), PE mouse IgG₁κ isotype control (Biolegend, USA), PE-Cy7 mouse IgG₁κ isotype control (Biolegend, USA), PerCP-vio700 mouse IgG₁ Isotype (Miltenyi, German), and APC mouse IgG₁κ isotype control (Biolegend, USA) 10 μl, respectively were intubated for 15 min in the dark room at room temperature, 500 μl RBC Lysis Buffer (Macs, German) was added into the tube. The mixture was centrifuged with 2,000 rpm for 5 min after another intubating for 15 min in the dark room at room temperature, then removed the supernatant and added 500 μl PBS fluid into the precipitate to resuspend cells which was analyzed by eight-color three-laser-MACSQuant Analyzer (Miltenyi Biotec, German) while data analysis performed using FlowJo software (Tree Star, USA).

Added 100 μl peripheral anticoagulant blood in healthy volunteer or sepsis patient into the tube with following anti-human fluorochrome-conjugated mAbs or specific isotype controls: PE mouse anti-human CD66b (Biolegend, USA), PE-Cy7 mouse anti-human CD10 (Biolegend, USA), PE mouse IgG₁κ isotype control (Biolegend, USA), PE-Cy7 mouse IgG₁κ isotype control (Biolegend, USA), PerCP-vio700 mouse IgG₁ Isotype (Miltenyi, German), and APC mouse IgG₁κ isotype control (Biolegend, USA) 10 μl, respectively. After intubating for 15 min in the dark room at room temperature, 500 μl RBC Lysis Buffer (Macs, German) was added into the tube. The mixture was centrifuged with 2,000 rpm for 5 min after another intubating for 15 min in the dark room at room temperature, then removed the supernatant and added 500 μl PBS fluid into the precipitate to resuspend cells which was analyzed by eight-color three-laser-MACSQuant Analyzer (Miltenyi Biotec, German) while data analysis was performed by using FlowJo software (Tree Star, USA).