Flow cytometry was performed by staining with Zombie Yellow viability dye, blocking with anti-CD16/32, and staining for 30 min at 4°C with the following anti-human antibodies: CD45 (BD:HI30), CD3 (BD:UCHT1), CD11b (Biolegend:M1-70), CD206 (Biolegend:15-2), CD163 (Biolegend:GHI/61), CD271 (Biolegend:ME20.4), PD-L1 (Biolegend:29E.2A3); or anti-mouse antibodies: CD45 (Biolegend:30-F-11), CD11b (Biolegend:M1-70), F4/80 (Biolegend:F4/80), Ly6G (Biolegend:1A8), Ly6C (Biolegend:HK1.4), IA/IE (BD:M5/114), CD206 (Biolegend:C068C2), PD-L1 (Biolegend:10F.9G2), PD-1 (BD:J43), IFN-γ (Biolegend:XMG1.2), CD3 (Tonbo:145-2C11), CD8 (Biolegend:YTS156.7.7), CD4 (BD:GK1.5), CD106 (Biolegend:429-MVCAM.A), EpCAM (Biolegend:G8.8), CD44 (Biolegend:IM7), Sca1 (Biolegend:D7). For IFN-γ, cells were pre-stimulated with PMA (20 ng/mL), Ionomycin (Sigma, 1 μg/mL) and Golgi stop (BD, 0.8 μL/106 cells) for 4 hr. Samples were subsequently run using BD FACS LSRII or sorted using BD FACS ARIA. Data were analyzed using FlowJo.
Pd l1
Manufactured by BioLegend
Sourced in United States, Germany
PD-L1 is a cell surface protein that plays a key role in regulating the immune system. It is expressed on various cell types, including tumor cells, and interacts with the PD-1 receptor on T cells, leading to the inhibition of T cell activation and immune responses. PD-L1 is a critical component in the immune checkpoint pathway and is a target of interest for immunotherapeutic approaches.
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Lab products found in correlation
Cd11b, by BioLegend (14 mentions)
F4 80, by BioLegend (13 mentions)
Cd206, by BioLegend (12 mentions)
Ifn γ, by BioLegend (9 mentions)
Cd62l, by BioLegend (8 mentions)
Hla dr, by BioLegend (5 mentions)
Lsrfortessa, by BD (5 mentions)
Tim 3, by BioLegend (5 mentions)
Cd163, by BioLegend (4 mentions)
Mhc 1, by BioLegend (4 mentions)
Tnf α, by BioLegend (4 mentions)
Pd l2, by BioLegend (4 mentions)
Cd103, by BioLegend (3 mentions)
Facscanto, by BD (3 mentions)
Bovine serum albumin (bsa), by Carl Roth (3 mentions)
Cd104, by BioLegend (3 mentions)
Facsaria 2, by BD (3 mentions)
Cd271, by BioLegend (2 mentions)
Epcam, by BioLegend (2 mentions)
Mhcii, by BioLegend (2 mentions)
75 protocols using pd l1
1
Multi-parameter Flow Cytometry Profiling
2
Flow Cytometric Immune Profiling
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Murine cells were incubated with a Ghost Dye Red 780 (Tonbo Biosciences) viability marker and anti-mouse CD16/32 (Tonbo Biosciences) in FACS buffer for 30 minutes on ice to block murine Fc receptors. Cells were then stained with the following fluorophore-conjugated anti-mouse monoclonal antibodies: CD45, CD3, CD4, CD8, ICOS [C398.4A], CD11c, CD11b, GR1 [RB6-8C5], F4-80, CD206 [C068C2], B7-1, MHC-I [34-1-2S & 28-8-6], MHC-II [M5/114.15.2], PD-1, Tim3, PD-L1, and B7x [HMH4-5G1] (all from Biolegend); and SPSYVYHQF/H-2Ld Alexa-647 conjugated tetramer [NIH Tetramer Core Facility]. All antibodies were stained for an additional 45 minutes on ice. Human cells were incubated with the Ghost Dye Red 780 viability marker for 30 minutes on ice and immediately stained with the following fluorophore-conjugated anti-human monoclonal antibodies: MHC-I, MHC-II, PD-L1, and B7x [MIH43] (all from Biolegend) on ice for 45 minutes.
3
Evaluating PD-L1 Downregulation by CARG-2020
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To examine the in vitro PD-L1 downregulation by CARG-2020, MC38 cells were stimulated with 20 ng/mL mouse IFN-γ and at the same time infected with two multiplicity of infection (MOI) of the indicated VLVs. After 26 h of stimulation/infection, cells were lifted and stained for PD-L1 (cat#124308, BioLegend), and analyzed for the percentage of PD-L1-positive and -negative cells by flow cytometry. For data analysis, we used GraphPad Prism software, version 9 (GraphPad Software, San Diego, CA). BHK21 cells stably expressing PD-L1 transgene were infected with PD-L1 shRNA VLV at 1 MOI infection. Cells collected by scraping and analyzed by Western blot probed with anti-PD-L1 (Bio X cell # BE0101), and anti-β-actin (Santa Cruz Biotechnology) antibodies. PD-L1 band densities were analyzed and normalized with actin loading control.
4
Imaging Tumor Microenvironment Immune Markers
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Frozen sections of the primary tumor and liver metastases were fixed with 4% paraformaldehyde in PBS and blocked with blocking solution (5% horse sera and 1% goat sera in PBS). Immunohistochemical staining of tissue sections was performed using antibodies against PD-L1 (BioLegend, San Diego, CA, USA), CD31 (BD Biosciences, San Jose, CA, USA), fibrinogen (Abcam, Cambridge, MA, USA), and various immune cells, such as CD8 (Invitrogen, Waltham, MA, USA), F4/80 (Abd Serotec, Raleigh, NC, USA), and CD45 (BD Pharmingen, San Diego, CA, USA). To image delivery of iv injected rat anti-PD-L1 IgG or isotype control IgG in tumors, the tissue sections were incubated with Alexa-fluor 647-labeled anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA). All images were acquired using laser scan confocal microscopy (Nikon Inc., Tokyo, Japan). ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the area fractions of positive staining on the captured fluorescence images [15 ]. All graphs are plotted based on the area fractions, and each symbol represents an individual imaging analysis.
5
Flow Cytometry Profiling of Immune Cells
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For membrane protein expression and intracellular staining, cells were washed and stained with Alexa-Fluor tagged antibodies. Fluorescence signals were measured with BD FACS Canto flow cytometer and BD Fortessa flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed with FACs DIVA software (BD Biosciences). Antibodies used included CD3, CD4, CD8, IFN-γ, CD45, and PD-L1 (Biolegend, San Diego, CA, USA). Therapeutic PD-L1 antibody and control antibodies were from Bio X Cell (West Lebanon, NH, USA).
6
Western Blot Analysis of PI3Kδ, p-AKT, PD-L1
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Cells were washed, scraped, and resuspended in lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Proteins were solubilized by sonication, separated by SDS-PAGE, and electroblotted onto polyvinylidenedifluoride membranes (Millipore). Membranes were blocked in Tris-buffered saline and Tween-20 solution containing 5% skim milk or bovine serum albumin and then probed with a primary antibody directed against PI3Kδ (Abcam), p-AKT (Cell Signaling Technology), PD-L1 (Biolegend, San Diego, CA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing and blocking, membranes were incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at dilutions of 1:10,000 for 1 h.
7
Analyzing SARS-CoV-2-specific T-cell responses
8
Comprehensive Antibody Panel for Immune Cell Analysis
9
Dissociating and Immunophenotyping huTGOs
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After 48 h of treatment, the cell co-cultures were incubated in Accutase (at 37 °C for 15 min) to dissociate huTGOs into single cells as previously published [30 (link)]. All cells were collected by centrifuging at 300× g for 5 min, then resuspended in 100 µL of diluted Zombie UV dye (1:100 in PBS) and stained for 15 min at RT. The cells were incubated at 4 °C for 30 min with fluorochrome-conjugated antibodies specific for CD8a, CD14, CD15, CD11b, CD33, CD137, EpCAM, granzyme B, Perforin, HLA-DR, and PD-L1 (1:100 dilution, all from BioLegend), diluted in 100 μL cell staining buffer. Cells were washed with cell staining buffer (BioLegend) and incubated with the Cytofix/Cytoperm Fixation/Permeabilization Buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at 4 °C. Cells were then washed and resuspended in 100 µL of cell staining buffer and stained at 4 °C for 30 min with fluorochrome-conjugated intracellular antibodies specific for perforin, IL-2 and interferon-gamma (IFN-g) (both from BioLegend) diluted in 100 µL cell staining buffer. Cells were washed, resuspended in 300 µL of cell staining buffer, filtered, and then analyzed on an LSRII system (BD Biosciences). An unstained cell sample and single stained beads for each antibody were used as gating controls. Data were analyzed using FlowJo software (BD Biosciences).
10
Flow Cytometry and Cell Sorting Protocols
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Flow cytometry was performed on Beckman Coulter CytoFlex and analyzed using FlowJo software (TreeStar). Mean fluorescence intensity (MFI) was calculated by genomic mean in FlowJo. Cell-sorting experiments were conducted on a BD Aria II. Staining was performed at 4°C in the presence of Fc block (Clone 2.4G2; BD) and FACS buffer (PBS, 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide). In the case of intracellular cytokine staining, brefeldin A (eBioscience) was added with peptide (10 nM SIINFEKL for OT-I cells) or TLR ligands (for BMDCs) for 4–8 h before staining with the intracellular staining kit (eBioscience). The following antibodies were all purchased from eBioscience unless otherwise indicated: anti-CD8α (53-6.7), IL-2 (JES6-5H4), CD69 (H1.2F3), TNF-α (MP6-XT22), CD45.1 (A20), CD44 (IM7), CD80 (16-10A1), IFN-α (RMMA-1), CD11C (N418), Clec9A (42D2), IFN-γ (XMG1.2), MHC II (M5-114.15.2), CD24 (M1-69), CD86 (GL1), PD-L1 (NIH5), CD127 (A7R34), XCR1 (ZET; BioLegend), CD103 (2E7), KLRG1 (2F1), IL-12p40 (C17.8), PD-1(J43), CD40 (IC40), SIINFEKL-peptide bound to H2-Kb (25.D1.16), LCMV gp33-H-2Db (gp33-41; MBL), and H-2Kb (AF6-88.5.5.3). MFI were calculated by genomic mean.
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