Anti cd45 apc cy7

Cells were immunostained with the following monoclonal anti-human antibodies: APC-H7 anti-CD45, pacific blue anti-CD3, PeCy7 anti-CD8, V450 anti-CD56, APC anti-IFN-γ, (BD Bioscience, San Jose, CA), APC Alexa Fluor 700 anti-CD4 (Beckman Coulter, Milan, Italy), Percp Cy 5.5 anti-TNF-α (eBioscience, Milan, Italy), APC anti-perforin, and PE anti-granzyme B (Invitrogen). Cells were immunostained with the following anti-mouse antibodies: APC-Cy7 anti-CD45, FITC anti-IFN-γ (BD Bioscience), PE anti-TNF-α, APC anti-perforin, and Pecy7 anti-granzyme B (eBioscience). In all experiments, appropriate isotype control IgGs (Becton Dickinson and eBioscience) and fluorescence minus one controls were used. All antibodies were used at 1:100 final dilution. For intracellular immunostaining, cells were fixed and permeabilized using staining buffer set and permeabilization buffer (both from eBioscience) according to the manufacturer's instruction. Cells were analyzed by flow cytometry (Gallios, Beckman Coulter, Indianapolis, IN).

To obtain BAL fluid for analysis, a sterile, 22-gauge catheter was inserted into exposed tracheal lumen of anesthetized mice. BAL fluid collected from one 0.8-ml aliquot of PBS per mouse was placed on ice and centrifuged. Supernatants were stored at –20°C; cell pellets were resuspended in PBS. Living cells were counted by staining with 0.4% trypan blue. Differential cell counts were obtained from cytospun cells stained with Diff-quik (American Scientific Products, McGaw Park, IL, USA). For further typing by analysis of surface protein expression, BAL cells were transferred into FACS buffer (PBS, 0.5% albumin, 0.1% sodium azide). Cells were divided and stained with either FITC anti-Gr1 (#RB6-865, UCSF Antibody Core Facility, San Francisco, CA, USA), PE anti-CD86 (553692, BD Pharmingen, San Diego, CA, USA), PE-Cy7 anti-CD11c (25-0114-82, eBioscience, San Diego, CA, USA), and APC anti-F4/89 (17-4801-82, eBioscience), or with FITC anti-CD3 (1530–02, Southern Biotech, Birmingham, AL, USA), PE anti-CD4 (553049, BD Pharmingen), PE-Cy7 anti-CD8 (552877, BD Pharmingen), APC anti NK1.1 (130-102-350, Miltenyi Biotec, San Diego, CA, USA), and APC-Cy7 anti-CD45 (557659, BD Pharmingen). Cells were stained for 30 min on ice, washed and then fixed in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) before analyzing using a Navios flow cytometer (Beckman Coulter, Brea, CA, USA).

The DSS (Cat no.160110) was purchased from the MP Biomedicals (Aurora, USA). The HSPA5 inhibitor HA15 (batch no. 1609402-14-3) was purchased from the MedChemExpress (New Jersey, USA). Dexamethasone sodium phosphate injection was obtained from the Western Pharmacy of Wuhan Union Hospital. ELISA kits, IL-10 (batch no. JYM0005Mo) and IL-6 (batch no. JYM0012Mo), were purchased from the ColorfulGene Biological Technology Company, Wuhan; TGF-β1 (batch no. E-EL-0162c) was purchased from the Elabscience (Wuhan, Hubei province). The anti-HSPA5 antibody (batch no. 11587-1-AP) was purchased from the Proteintech Group (Chicago, IL). The anti-HSPA1A antibody (batch no. PA534772) was purchased from the Thermo Company (Wyman Street, Waltham, MA), and the anti-CHIP antibody (batch no. 2080S) was purchased from the CST Company (Danvers, MA). FVS 510 (batch no. 564406), PE-Cy7-anti-CD4 (batch no. 552775), APC-Cy7-anti-CD45 (batch no. 557659), and PE-anti-FOXP3 (batch no. 563101) were purchased from the BD Biosciences (San Jose, CA).

For fluorescence activated cell sorting (FACS), single cell suspension was prepared as described previously (22 (link)) from fresh COAD specimens and incubated with Human BD Fc Block (BD Bioscience). Then, the samples were stained with APCcy7 anti-CD45 (561863, BD bioscience), FITC anti-CD14 (555397, BD bioscience), and BV421 anti-CSF-1R (565347, BD bioscience) antibodies at 4°C for 30 min in dark. After washing cells 3 times with PBS, the cells were resuspended with staining buffer, and subjected to cell sorting using a BD FACS Aria3 flow cytometry. CSF-1R^low TAMs (CD45⁺ CD14⁺ CSF-1R^low) and CSF-1R^high TAMs (CD45⁺ CD14⁺ CSF-1R^high) were obtained from single-cell mixed suspension by flow cell sorting. Total RNA was isolated from the sorted samples using Qiagen RNeasy kit and subjected to cDNA library preparation using Smart-seq2 (32 (link)) scheme. The sequencing was performed using Illumina Novaseq6000 platform (33 (link)).

PBMCs were incubated with immunogenic peptide pools for nine days with three cycles of stimulations. After stimulation, PBMCs were washed and stained with anti CD3 AF700, anti CD8 APC, and Propidium iodide (PI, BD). Peptide specific live T cells (CD3⁺CD8⁺PI⁻) were FACS isolated for organoid killing assay. Organoids were dissociated to single cells with Tryple Express, labeled with CFSE (BD), and cocultured in at least triplicate with sorted T cells at a 10:1 effector:target ratio in T cell culture medium. To analyze the activity of ICIs, 5 µg mL⁻¹ PD‐1 inhibitor and/or 10 µg mL⁻¹ CTLA‐4 inhibitor was added during the progress of stimulation and killing assay. Each organoid killing assay was repeated twice. After 3 days of coculture, organoids and T cells were collected. Cells were washed in FACS buffer and stained with anti CD45 APC‐Cy7, Annexin V PE, 7‐AAD (BD) in Annexin V buffer (BD) for 15 min at RT. The apoptosis of organoids labeled with CFSE were analyzed with flow cytometer Canto II (BD) or CytoFLEX (Beckman). FITC⁺CD45 APC‐Cy7⁻ was used to detect organoids and Annexin V⁻7‐AAD⁻ was used to define live cells.

Human peripheral blood mononuclear cells from the G-CSF-treated healthy volunteer were stained in an AutoMACS Running Buffer in 4°C with the following antibodies: cocktail of antibodies for hematopoietic lineage markers (Lin) (anti-CD2-FITC (#555326, clone RPA2.10), anti-CD3-FITC (#555332, clone UCHT1), anti-CD14-FITC (#345784, clone M5E2), anti-CD16-FITC (#555406, clone 3G8), anti-CD19-FITC (#555412, clone HIB19), anti-CD24-FITC (#555427, clone ML5), anti-CD56-FITC (#345811, clone NCAM16.2), anti-CD66b-FITC (#555724, clone G10F5), and anti-CD235a-FITC (#559943, clone GA-R2(HIR2), all 1 : 200) all from BD Pharmingen), anti-CD45-APC-Cy7 (#304014, clone 2D1, BD Biosciences, 1 : 50), anti-CD34-APC or anti-CD34-FITC (#555824, #555821, clone 581, BD Pharmingen, 1 : 50), anti-CD133-PE (#130-080-801, clone AC133, Miltenyi Biotec, 1 : 50), anti-CD105-PE (#323206, clone 43A3, BioLegend, 1 : 50), anti-CD90-APC (#559869, clone 5E10, BioLegend), anti-CD181-APC (#551080, clone 5A12, BD Pharmingen), anti-CD184(CXCR4)-APC (#555976, clone 12G5, BioLegend, 1 : 50), anti-CD11b-APC (#101211, clone M1/70, BioLegend, 1 : 50), and 10 μg/mL Hoechst 33342 (B2261-25MG, Sigma Aldrich). Stained cells were then analyzed on a BD LSR II flow cytometer using BD FACS Diva software (Becton Dickinson). Controls for gating are shown in Supplementary Figure 1.

All the reagents, unless differently specified, were obtained from Sigma Aldrich (Milan, Italy). Antibodies against NF-kB (C-20, sc-37), PPARα (H-98, sc-9000), β-actin (I-19, sc-1616), and monoclonal antibodies against PPARγ (E-8, sc-7273) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies anti-CD34 PE were from Miltenyi-Biotech; antibodies anti-CD90 FITC, anti-CD105 APC, anti-CD29 PerCP Cy5-5, anti-CD31 FITC, anti-CD44 PerCP Cy5-5, anti-CD45 APC-Cy7 and anti-CD14 BD HORIZON V500 were from BD Pharmingen. All cell culture materials were purchased from Gibco (Invitrogen, Milan, Italy).