For flow cytometry based quantification in Figure S1 , PD-L1 and CD80 were stained by PE anti-PD-L1 (eBioscience, 14-5983-82) and PE anti-CD80 (Biolegend, 305208) and their expression levels were quantified using the QUANTUM R-PE MESF kit (Bangs Laboratories Inc, 827), following manufacturer’s instructions. For immunoblot-based quantifications, total cell lysates were subjected to SDS-PAGE, transfected to a nitrocellulose membrane. Afterward, PD-L1 was probed by PE anti-PD-L1 (eBioscience, 14–598382) and detected by a Typhoon 5 Biomolecular Imager; CD80 was sequentially probed by anti-CD80 (Novus Biologicals, NBP2–25255) and DyLight488 anti-mouse IgG (Biolegend, 405310), then detected by Typhoon 5 Biomolecular Imager. Molecular densities were calculated assuming the following diameters:13 μm for Raji B cells (Hui et al., 2017 (link)) and 12.5 μm for DCs (Dumortier et al., 2005 (link)).
Anti pd l1 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-PD-L1-PE is a fluorescently-labeled antibody that binds to the PD-L1 protein, which is expressed on the surface of certain cells. It can be used for the detection and quantification of PD-L1 expression in laboratory research applications.
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Lab products found in correlation
Anti cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Anti cd8 fitc, by Thermo Fisher Scientific (3 mentions)
Anti cd80 fitc, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by R&D Systems (2 mentions)
Anti pd 1 pe, by Thermo Fisher Scientific (2 mentions)
Anti gr1 fitc, by Thermo Fisher Scientific (2 mentions)
Anti cd11b pe, by Thermo Fisher Scientific (2 mentions)
Anti cd80 pe, by BioLegend (2 mentions)
Lsrii cytometer, by BD (1 mentions)
Anti pd 1 fitc, by Thermo Fisher Scientific (1 mentions)
Anti t bet bv711, by BioLegend (1 mentions)
Anti cd45ra ecd, by Beckman Coulter (1 mentions)
Anti cd27 apc cy7, by BioLegend (1 mentions)
Anti cd3 alexa 700, by BD (1 mentions)
Anti cd20 ecd, by Beckman Coulter (1 mentions)
Anti cd8 pe cy5, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua stain, by Thermo Fisher Scientific (1 mentions)
Anti eomes efluor 660, by Thermo Fisher Scientific (1 mentions)
Anti cd86 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd11c alexa700, by Thermo Fisher Scientific (1 mentions)
15 protocols using anti pd l1 pe
1
Quantification of PD-L1 and CD80 Levels
2
Comprehensive PBMC Immunophenotyping Protocol
3
Immune Checkpoint Expression Profiling
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Post 72 hours transfection, adherent cells were digested and then resuspended in PBS for subsequent staining procedures. The cell suspensions were incubated with the following fluorescently conjugated antibodies for 30 minutes: anti-PD-L1-PE (Invitrogen, 12–5982-81), anti-PD-L2-PE (Biolegend, 329606), anti-CD70-FITC (Biolegend, 355106), anti-CD47-AF700 (Biolegend, 323126), and anti-Galectin-9-FITC (Biolegend, 348912). After the staining, cells were washed once with PBS. Subsequently, 7AAD (Biolegend, 420403) was added to discriminate live cells. Flow cytometry analysis was conducted using the MA900 flow cytometer (SONY, Japan). The recorded data were analyzed using FlowJo software (Treestar, Ashland, USA).
4
PD-L1 Expression Analysis in Septic Mice
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Flow cytometry was performed to detect the PD-L1 expression level in the PD-L1 humanized mice after CLP surgery. Blood samples were collected by heart puncture and anticoagulated in a BD Vacutainer (BD, Franklin Lakes, New Jersey, U.S.A.) 24 h after surgery. The white blood cells were stained with anti-CD3-APC, anti-CD11b-FITC, anti-Ly6C-APC, anti-Ly6G-APC, and anti-PD-L1-PE antibodies (purchased from eBioscience, San Jose, California, U.S.A.). Flow cytometry assays were performed with a FACSCalibur Flow Cytometer (BD Biosciences, Heidelberg, Germany) and the data were analyzed with FlowJo 7.6 software (Tree Star, Ashland, Oregon, U.S.A.). The level of PD-L1 expression was shown as the percentage of PD-L1-positive cells.
5
Multi-parameter Immune Cell Profiling
6
Investigating PD-L1 Regulation and Signaling
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SA-49 was synthesized as described previously and dissolved in DMSO [21 (link)]. LY294002, Go6976, 5Z-7-Oxozeaenol and Torin1 were purchased from Selleck (Beijing, China). Cycloheximide (CHX), MG132, and Bafilomycin (Baf) were purchased from Sigma (St. Louis, MO, USA). Antibodies against PD-L1, TFEB, MITF, H3, PKCα, p-GSK3β (Ser9), cleaved caspase 9 and 3 were purchased from Cell Signaling (Danvers, MA, USA). Anti-GSK3β and GAPDH antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-PD-L1-PE, IgG-PE and FoxP3 antibodies were purchased from eBioscience (San Diego, CA, USA). Antibodies against p-PKCα (T638), CD3 and Ki67 were obtained from Abcam (Cambridge, MA, USA). The probes LysoTracker and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Human PD-1 Fc recombinant protein and IL-2 were purchased from R&D Systems (Minneapolis, MN, USA).
7
Exosome Protein Detection by Flow Cytometry
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The on-bead flow cytometry was used for detection of proteins carried by exosomes as previously reported20 -22 and described in Supplemental Methods . The following detection Abs were used: anti-PD-L1 PE (12–5983-42, e-Bioscience) or anti-PD-1 PE (12–2799-42, e-Bioscience), anti-CTLA4-APC (349908, Biolegend), anti-CD15s-PE (sc-32243, Santa Cruz), anti-CD3-PE (12–0037-42, e-Bioscience). Isotype control Abs were used in all experiments.
Detection of exosome cargo components was performed using the Gallios flow cytometer equipped with Kaluza 1.0 software (Beckman Coulter, Krefeld, Germany). Samples were run for 2 min and around 10,000 events were acquired. Gates were set on the bead fraction visible in the forward/sideward light scatter. Reproducibility of this flow cytometry-based detection assay was established as described inSupplemental Methods . Flow cytometry results of a representative experiment are shown in Supplemental Figure 3.
Detection of exosome cargo components was performed using the Gallios flow cytometer equipped with Kaluza 1.0 software (Beckman Coulter, Krefeld, Germany). Samples were run for 2 min and around 10,000 events were acquired. Gates were set on the bead fraction visible in the forward/sideward light scatter. Reproducibility of this flow cytometry-based detection assay was established as described in
8
Multiparametric Immune Profiling Protocol
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Anti-mouse monoclonal antibodies directly conjugated to fluorophores against the following targets of interest were used: F4/80, PD-L1, and Foxp3 (eBioscience, Germany); CD4, CD3e, CD44, CD8a, CD62L, CD45R, CD11b, CD11c, Gr-1, NK1.1, CD25, Ly6C, and PD-1 (BD Bioscience, Germany). Fc receptor binding inhibitor (anti-mouse CD16/CD32) was purchased from eBioscience (Germany). Anti-human antibodies included anti-PD-L1-PE and purified anti-PD-L1 blocking antibody (both from eBioscience, Germany). For western blot, anti-phospho-ERK42/44, anti-phospho-p38 and anti-phospho STAT3 along with their reference antibodies, anti-ERK42/44, anti-p38, and anti-STAT3 antibody (all from Cell Signaling Technology, USA), were used. Signal transduction inhibitors: UO126 (MEK1/2 inhibitor, Cell Signaling Technology, USA), SB203580 (p38 MAPK inhibitor, Cell Signaling Technology, USA), LY294002 (PI3K inhibitor), CAS457081-03-7 (Jak inhibitor 1, Calbiochem, Germany) and Cucurbitacin/JSI-124 (STAT3 inhibitor, Calbiochem, Germany). Cytokines used included IL-4, GM-CSF, TNF-α, IL-6, IL-1β, PGE2 (Promokine, Immunotools, Strathmann, Germany) and IFNα (R&D; IntronA, Interferon alfa-2b, Schering-Plough; Germany).
9
Flow Cytometric Analysis of Tumor-Infiltrating Immune Cells
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Splenocytes or lymphocytes were stained using anti-PD-L1 PE, anti-PD-1 PE, anti-CD4 FITC, anti-CD8-FITC and anti-CD107a-PE (eBioscience, San Diego, CA) as indicated by the manufacturer. Specific mAb isotype-matched control was also used. Sera from naïve and mice cured by combined anti-PD-1 and anti-CD4 mAb therapy were used to assess reactivity against Neuro2apc or NXS2pc cells at dilutions ranging from 1:10 to 1:10,000. A FITC-conjugated goat anti-mouse antiserum (Jackson Labs,West Grove, PA) was used as a second-step reagent. Anti-GD2 mAb supernatant (M36.1-S2a) (ATCC) was used as positive control.
Tumors from Neuro2a- and NXS2 bearing mice were dissociated mechanically and immune cells infiltrates were stained with anti-CD4 FITC, anti-CD25 APC, anti-LAG-3 PE, anti-Gr1 FITC and anti-CD11b PE (eBioscience, San Diego, CA) as indicated by the manufacturer. Samples were analyzed by a FACScan or FACSCalibur analyzer (Becton Dickinson).
Tumors from Neuro2a- and NXS2 bearing mice were dissociated mechanically and immune cells infiltrates were stained with anti-CD4 FITC, anti-CD25 APC, anti-LAG-3 PE, anti-Gr1 FITC and anti-CD11b PE (eBioscience, San Diego, CA) as indicated by the manufacturer. Samples were analyzed by a FACScan or FACSCalibur analyzer (Becton Dickinson).
10
Murine Myeloid-Derived Suppressor Cell Characterization
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RPMI 1640, DMEM, FBS, and antibiotics were obtained from Life Technologies. Recombinant murine GM-CSF, anti–TGFB1-APC, and IL-2 were obtained from R&D Systems. The following antibodies were purchased from eBioscience: anti-Gr1 FITC, anti-Gr1 PE, anti-F4/80+ PE, anti-Cd11b+ PE, anti-CD11c+ APC, anti–PD-L1 PE, anti–PD-L1 FITC, anti–PD-L1 APC, anti–PD-L2 PE, anti-CD80 FITC, anti-CD86 FITC, anti–PD-1 APC, anti–CTLA-4 APC, anti-CD4 FITC, anti-CD8 FITC, anti–IFN-γ PE-cy7, anti-IL6 FITC, anti-IL10 APC, anti-IL12p70 PE, and functional grade anti–PD-L1 (MIH5) neutralizing antibody. For blocking, control antibody (IgG; rat IgG2b K Isotype Control Functional Grade Purified; eBioscience), anti–mouse IL-6 Functional Grade Purified neutralizing antibody (eBioscience), or anti–mouse IL-10 Functional Grade Purified neutralizing antibody (eBioscience) were used.
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