Flow cytometry were carried out using antibodies listed as follows: CD3e [BD Biosciences, 561824 (PE)], CD4 [Thermo Fisher Scientific, 11-0042-82 (FITC)], CD8a [BioLegend, 100706 (FITC)], CD11b [BD Biosciences, 561098 (PE/Cy7)], CD11c [BioLegend, 117353 (BV510)], CD19 [BD Biosciences, 561736 (PE)], CD25 [Thermo Fisher Scientific, 17-0251-82 (APC)], CD45 [BD Biosciences, 561037 (APC-Cy7)], CD49b [BD Biosciences, 561066 (PE)], CD80 [BioLegend, 104714 (APC)], CD274 [BioLegend, 124315 (BV421)], Ly-6G [BD Biosciences, 561104 (PE)], MHC-II (I-A/I-E) [BD Biosciences, 562363 (PerCP-Cy5.5)], and FOXP3 [BioLegend, 126419 (BV421)]. To determine the relative expression of surface markers, cells were stained in FACS buffer for 30 min at 4 °C. For intracellular staining, FOXP3 staining buffer kit (Thermo Fisher Scientific, Waltham, MA) was used following the manufacturer's instructions. Flow cytometry analyses were conducted on a LSR II instrument (BD Biosciences, Mountain View, CA). Results were obtained from the FACSDiva V 7.0 software (BD Biosciences, Mountain View, CA), and all data were analyzed by FlowJo Version 10.0 software. As for FACS, splenic mononuclear cells were isolated, prepared, and stained as described above, followed by sorting of mregDCs as well as non-mregDCs using BD FACSAria flow cytometer (BD Biosciences, Mountain View, CA).
Cd274
Manufactured by BioLegend
Sourced in United States
CD274 is a cell surface protein that functions as a ligand for the PD-1 receptor. It plays a role in regulating T cell activation and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 intracellular staining kit, by Thermo Fisher Scientific (2 mentions)
Cd11b, by BioLegend (2 mentions)
F4 80, by BioLegend (2 mentions)
Cd103, by BioLegend (2 mentions)
Hla dr, by BioLegend (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (1 mentions)
Mhcii, by BD (1 mentions)
Facs diva v7, by BD (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Cd11b, by BD (1 mentions)
Lsrii instrument, by BD (1 mentions)
Facsverse instrument, by BD (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Bay60 6583, by Corning (1 mentions)
Cd146, by BioLegend (1 mentions)
Cd166, by BioLegend (1 mentions)
Hla g, by BioLegend (1 mentions)
Facsaria 3, by BD (1 mentions)
Cd19 fitc, by BioLegend (1 mentions)
9 protocols using cd274
1
Comprehensive Immune Cell Profiling by Flow Cytometry
2
Multicolor Flow Cytometry Analysis
3
Quantifying Immune Checkpoint Expression in NSCLC
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The surface expression of CD326, PD-L1, IL-1R1, IFNGR1, and IFNGR2 in NSCLC cells, and the surface expression of CD11b in THP-1-derived macrophages was quantified by flow cytometry. Cells were stained with antibodies to CD326 (BioLegend, #324306), CD274 (BioLegend, #329708), IL-1R1 (R&D Systems, #FAB269P), IFNGR1 (R&D Systems, #FAB673P), IFNGR2 (R&D Systems, #FAB773A), CD11b (eBioscience, #11-0118-42), allophycocyanin-conjugated mouse IgG2b isotype control (BioLegend, #400322), phycoerythrin-conjugated goat IgG isotype control (BioLegend, #403004), phycoerythrin-conjugated mouse IgG1 isotype control (BioLegend, #400114), allophycocyanin-conjugated goat IgG isotype control (R&D Systems, #IC108A), or fluorescein isothiocyanate-conjugated mouse IgG1 isotype control (BioLegend #400109). For the functional analysis of PBMCs co-cultured with tumor cells, PBMCs were stained with antibody to CD3 (BioLegend, #344833), CD8 (BioLegend, #344711), CD4 (BioLegend, #564420), PD-1 (BioLegend, #329905), or Tim3 (BioLeged, #345011). All samples were analyzed by flow cytometry using a FACSVerse instrument (BD Biosciences). The data were analyzed by Flowjo (v10.5.3) Software. The relative mean fluorescence intensity (MFI) ratio was calculated as the MFI for PD-L1, IL-1R1, IFNGR1, or IFNGR2 divided by that for the corresponding isotype control.
4
Inhibition of A2BR and PD-L1 in CAL27 Cells
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CD274 (PD-L1-neutralizing antibody) was purchased from BioLegend (USA), BAY60-6583 (A2BR agonist) and MRS-1706 (A2BR inverse agonist) were purchased from MedChemExpress (USA), and PDTC (NF- κB inhibitor) was purchased from Abcam (UK). CAL27 cells were seeded at a density of 1 ×105/mL in 6 well plate (Corning, Corning, NY, USA) and treated with 100 nM, 1 µM, or 10 µM BAY60-6583 for 24 h or treated with 4 µg/mL PDTC 1 h before BAY60-6583 treatment. Additionally, CAL27 cells were seeded at a density of 1 ×105/mL and treated with 4 µg/mL CD274 and/or 1 µM MRS-1706 for 24 h, as the control group, MRS-1706 group, PD-L1 Nab group and PD-L1 Nab+MRS group. The media was changed and supernatants were collected after 24 h of incubation.
5
Immunophenotyping of Extracellular Vesicles
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Anti‐CD63 magnetic beads (Dynabeads 10606D, Thermo Fisher Scientific; 15 μl containing 1.5 × 105 beads) were incubated with samples containing 107–108 EV particles in a volume of 50 μl bovine serum albumin (BSA), 1 mg/ml in PBS (BSA‐PBS) overnight at 4°C with gentle shaking. Preliminary experiments indicated that these conditions captured >85% of EV particles measured by TRPS or fluorescent‐labeled EVs (data not shown). The samples were rinsed 3× in PBS by magnetic capture and then incubated in a volume of 50 μl PBS‐BSA containing fluorescent antibodies to one or combinations of the following markers: CD81, CD9, CD29, CD13, CD90, CD36, HLAG, HLA‐DR, CD34, CD146, CD107a, CD274, CD166, CD44, CD74, or CD59 (BioLegend, CA) for 2 hours at room temperature. After 3× rinsing in PBS the beads were analyzed by flow cytometry using a BD FACS Aria III instrument. A majority of beads with mean fluorescence greater than that of the isotype control antibody was considered indication of presence of the targeted protein (Supporting Information Fig. S1). Triplicate analyses were carried out for all samples.
6
Characterization of Immune Checkpoint Markers
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Purified murine peripheral blood mononuclear cells or tail vein blood were incubated with fluorochrome-labelled rat anti-mouse antibodies directed to the following antigens were used: PDCD1 [phycoerythrin (PE), clone RPM1-30], LAG3 (PE, clone C9B7W), CD274 (PE, clone 10F.9G2) or the appropriate isotype controls, CD3 (fluorescein isothiocyanate, FITC), CD5 (PE-cyanin 5.1, PC5), CD8 (Biotin), CD19 (FITC), (all from Biolegend, San Diego, CA, USA) and streptavidin (Texas Red) (BD Biosciences, San Jose, CA, USA). Erythrocytes were lysed using BD fluorescence-activated cell sorting (FACS) Lysing solution (BD Biosciences). Cells were then resuspended in phosphate-buffered saline and analysed using an FC500 or Gallios flow cytometer and CXP1.0 software or Gallios Cytometer Software 1.1 (Beckman Coulter, Indianapolis, IN, USA). Results are calculated as percent positive cells (PDCD1 and LAG3) or mean fluorescence intensity ratio (MFIR; CD274 vs isotype control) as indicated.
7
Comprehensive Immune Cell Phenotyping
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Antibodies used for immunostaining were against CD69 (biolegend 104545), CD25 (biolegend 102041), NK‐1.1 (biolegend 108753), CD3 (biolegend 100232), CD4 (biolegend 100423), CD8a (BD biosciences 566410), FoxP3 (biolegend 126404), CD45 (biolegend 103112), Ly‐6C (biolegend 128032), Ly‐6G (biolegend 127633), CD274 (biolegend 124331), F4/80 (biolegend 123110), CD11c (BD biosciences 566504), CD11b (biolegend 101217), CD86 (biolegend 105037), and CD103 (biolegend 562722). FoxP3 intracellular staining was carried out using FoxP3 intracellular staining kit (Thermo 00‐5523‐00) following manufacture protocol. Immunostained cells were run on an LSR Fortessa HTS with FACSDIVA software and analyzed using FlowJo V10.5.3.
8
Multiparameter Flow Cytometry Analysis
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All fluorochrome‐conjugated anti‐human antibodies against CD45 (Cat: 304062), CD3 (Cat: 300306), CD4 (Cat: 317444; Cat: 301051), CD8 (Cat: 300539), LAG3 (Cat: 369206), PD1 (Cat: 329918), NKG2D (Cat: 320808), HLA‐DR (Cat: 307604), CD274 (Cat: 329708), MICA/MICB (Cat: 320906), Annexin V (AV) (Cat: 422201), Human TruStain FcX™ (Cat: 422302) were purchased from Biolegend company. Data were collected using the Aria II Flow Cytometer (BD Biosciences), Attune NxT (Thermo), or Cytoflex (Beckman) and analyzed by FlowJo Software V10.
9
Assessing IFN-γ-Induced Checkpoint Modulation
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SJSA-1 cells were incubated for 24 h with 50ng/mL recombinant human IFN-γ (Cat No. 300-02, Peprotech), then the media was replaced with media containing 50 μg/mL of SBT-100 and incubated for 48 h. Cells were then harvested and stained with the following antibodies: CD276 (Clone MIH-42, Biolegend Cat. No. 351005, San Diego, CA, USA), CD274 (Clone 29E.2A3, Biolegend Cat. No. 329713), and CD200 (Clone OX-104, Biolegend Cat. No. 329217). Upon staining, samples were run through a BD Celesta Flow Cytometer, and data were analyzed using FlowJo version 10.2 software (BD Biosciences, Ashland, OR, USA).
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