Aqua dead cell stain kit

Dulbecco’s modified Eagle’s medium (DMEM), PBS, penicillin/streptomycin, l-glutamine, fetal bovine serum (FBS), Aqua Dead Cell Stain Kit, Lipofectamine 2000, TRIzol, and collagenase type IV were purchased from Thermo Fisher Scientific (Waltham, MA, United States). The RBC lysis buffer was purchased from Solarbio (Beijing, China). siRNA-targeting mouse CD47 mRNA (antisense strand, 5’-UGGUGAAAGAGGU-CAUUCCdTdT-3’) and negative control siRNA with a scrambled sequence (antisense strand, 5’-ACGUGACACGUUCGGAGAAdTdT-3’) were synthesized by Suzhou Biosyntech Co. Ltd. (Suzhou, China). Anti-CD47 antibody was purchased from Santa Cruz Biotechnology (Texas, United States). The Click-iT Plus TUNEL Assay was performed for in situ apoptosis detection; the Alexa Fluor 647 dye was purchased from Thermo Fisher Scientific (MA, United States).

The colonic tissues were cut into small pieces by scissors, and then processed in a digestion buffer and Miltenyi gentleMACS Dissociator following by instructions. Homogenized intestinal tissues were passed through a 40-μm nylon mesh to get a single-cell suspension. Cells were stained with F4/80, CD11b and CD86, CD11c and CD206 antibodies (eBioscience, San Diego, USA) and incubated for 20 min at 4°C in the dark. Macrophages were identified with CD11b+ F4/80+ and CD86+ macrophages were identified with CD11b+ F4/80+ CD86+. After washing, macrophage cell subsets were collected and flow cytometrically analyzed (FACScalibur Flow Cytometer, Becton-Dickinson Immunocytometry Systems, San Jose, USA). Cell viability was preformed using Aqua Dead Cell Stain Kit (L34965, Thermo Fisher Scientific, USA). The percentage of cells in live/singlets gate was evaluated by the number of live cells to get an absolute live-cell count. At least 10, 000 live events were accumulated through forward- and side-scatter parameters. Cell sorting was processed on a FACSAria II instrument (BD Biosciences, San Jose, USA) with the same configuration as the LSR II. Cytospins were prepared from the sorted cells and cell populations were identified using a sequential gating strategy. Data were analyzed by using FlowJo software (version 7.01, Tree Star Software, San Carlos, USA).

SV40-fibroblasts were incubated with the PE-Dazzle-594-PD-L1 (CD274) antibody (clone 29E.2A3, #329732, BioLegend), or the corresponding isotype (#400358, BioLegend). For pSTAT1 staining, the cells were starved overnight in DMEM-1% FBS. For intracellular staining, 10⁶ cells were washed with PBS-2% FBS- 2 mM EDTA buffer, fixed by incubation for 10 minutes at 37°C with Fix Buffer I (#557870, Beckton Dickinson,) and permeabilized by incubation for 20 minutes at 4°C with Phosflow Perm Buffer III (#558050, Beckton Dickinson). Cells were then incubated with PE-coupled STAT1 (clone 1, #558537, Beckton Dickinson) or PE- or AF647-conjugated anti-pSTAT1 antibody (clone 4a, # 612564 or #612597, Beckton Dickinson), the corresponding isotype (#554680 and #565363, respectively, Beckton Dickinson), or unconjugated IRF1 (clone D5E4, #8478, Cell Signaling) or the corresponding isotype, for detection with PE-conjugated goat anti-rabbit antibody (#A10542, Thermo Fisher Scientific). All non-fibroblastic cells were also stained with the Aqua Dead Cell Stain kit (#L34957, Thermo Fisher Scientific). Cells were acquired on a Beckman Coulter Gallios flow cytometer and analyzed with FlowJo Software.

Flowcytometry was performed using same materials and methods as described earlier.21 (link) Pulmonary single cell suspension was prepared using Lung dissociation Kit (130‐095‐927, Miltenyi Biotec) according to manufacturer instructions. The single‐cell suspension was then used for two different staining panels targeting myeloid or lymphoid populations. Viability staining was performed using Aqua Dead Cell Stain Kit (L34965, Thermo Fisher) or Near‐IR Dead Cell Stain Kit (L34975, Thermo Fisher) according to the manufacturer's instructions, followed by 10‐minute FC blocking. Myeloid or Lymphoid antibody mixes (Table 2) were applied for 30 minutes in 4°C prior to flow cytometry analysis. Figures S6 and S7 demonstrate gating strategies used in this study.

Human peripheral blood mononuclear cells (PBMCs) from a healthy donor were isolated from whole blood by Ficoll- Hypaque density centrifugation (Amersham-Pharmacia- Biotech). The cells were counted and plated at 2 × 10⁶ cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 μL of RPMI (GibcoBRL, Invitrogen) supplemented with 10% fetal bovine serum (GibcoBRL, Invitrogen), or 100 μL of RPMI supplemented with 1:10 serafrom patients or controls. PBMCs were left unstimulated or were stimulated with 10 ng/μL of rhIL-3 or GM-CSF or 50 ng/μL of rhIL-3 (Miltenyi-Biotec) for 15 min at 37°C. Thereafter, cells were fixed and permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with antibodies anti CD14-Pacific Blue and anti CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific). STAT5 phosphorylation (p-STAT5) levels were assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).