Pd l1 clone 10f 9g2

Manufactured by BioLegend

The PD-L1 (clone 10F.9G2) is a monoclonal antibody that binds to the PD-L1 (Programmed Death-Ligand 1) protein. PD-L1 is a cell surface protein that plays a role in the regulation of the immune response.

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Lab products found in correlation

Cd45 clone 30 f11, by BioLegend (3 mentions) Cd11c clone n418, by BioLegend (2 mentions) Cd11b clone m1 70, by BioLegend (2 mentions) Nk1.1 clone pk136, by BioLegend (2 mentions) Cd80 clone 16 10a1, by BioLegend (2 mentions) H 2kb clone af6 88.5, by BioLegend (2 mentions) Anti mouse cd45.2 clone 104, by BioLegend (2 mentions) Ultracomp ebeads, by Thermo Fisher Scientific (1 mentions) Cd16 32 fc block clone 2.4g2, by BD (1 mentions) Kaluza analysis software, by Beckman Coulter (1 mentions) Counting beads, by Spherotech (1 mentions) Mhc 1, by BioLegend (1 mentions) Anti cd16 cd32 fc block, by BD (1 mentions) Pe labeled h 2kb siinfekl pentamer, by ProImmune (1 mentions) Live dead fixable blue dead cell stain, by Thermo Fisher Scientific (1 mentions) F4 80 clone bm8, by BioLegend (1 mentions) Zombie aqua, by BioLegend (1 mentions) Cd86 clone gl 1, by BioLegend (1 mentions) Facscalibur, by BD (1 mentions) Fortessa, by BD (1 mentions)

7 protocols using pd l1 clone 10f 9g2

Comprehensive Tumor Cell Phenotyping

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Totally, 1.5 million cells from tumors in mTmG mice were stained following the BD Cytofix/Cytoperm Fixation/Permeabilization microplate staining protocol (BD Biosciences), using the following antibodies: CD16/32 Fc Block (clone 2.4G2, BD Biosciences cat. 553142, 1:100), CD45 (clone 30-F11, BioLegend cat. 103127, 1:350), PD-L1 (clone 10F.9G2, BioLegend cat. 124313, 1:100), CD11b (clone M1/70, BioLegend cat. 101223, 1:100), CD11c (clone N418, BioLegend cat. 117323, 1:100). Cell lines were stained with PD-L1 (clone 10F.9G2, BioLegend cat. 124307, 1:100). Dead cells were excluded using Ghost Dye Violet 510 Dead Cell Stain Kit (Tonbo Biosciences) according to manufacturer protocol, and compensation was calibrated with single-color controls on Ultracomp eBeads (Invitrogen). Cells were quantified using a Gallios 561 cytometer (Beckman Coulter) and analyzed using Kaluza analysis software (Beckman Coulter) with fluorescence-minus-one controls to verify gating.

Strait A.A., Woolaver R.A., Hall S.C., Young C.D., Karam S.D., Jimeno A., Lan Y., Raben D., Wang J.H, & Wang X.J. (2021). Distinct immune microenvironment profiles of therapeutic responders emerge in combined TGFβ/PD-L1 blockade-treated squamous cell carcinoma. Communications Biology, 4, 1005.

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Comprehensive Tumor Immunophenotyping by FACS

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FACS analysis was performed using 200-μL tumor or splenocyte single-cell suspensions per antibody panel. Cells were first incubated at 4°C for 30 minutes in PBS with anti-CD16/CD32 Fc block (BD Biosciences) at 1: 100 and live/death staining (Zombie Aqua, BioLegend) at 1: 1,000, or Live/Dead fixable blue dead cell stain (Invitrogen). Next, cells were washed and stained at 4°C for 30 minutes in PBS with antibodies against CD45 (clone 30-F11), B220 (clone RA3–6B2), CD3 (clone 17A2), CD8 (clone 53–6.7), MHC-I (clone AF6–88.5), PD-1 (clone RPM1–30), PD-L1 (clone 10F.9G2), NK1.1 (clone PK136), CD11b (clone M1/70), Ly6G (clone 1A8) and F4/80 (clone BM8), all from BioLegend, at 1 μg/mL. PE-labeled H-2Kb - SIINFEKL pentamer (ProImmune) was used at 2.5 μL per staining. In some sets of experiments, absolute cell numbers were calculated by adding 10 μL counting beads (1 × 10⁶ beads/mL, Spherotech) to each sample before acquisition.

Veerman R.E., Akpinar G.G., Offens A., Steiner L., Larssen P., Lundqvist A., Karlsson M.C, & Gabrielsson S. (2023). Antigen-Loaded Extracellular Vesicles Induce Responsiveness to Anti–PD-1 and Anti–PD-L1 Treatment in a Checkpoint Refractory Melanoma Model. Cancer Immunology Research, 11(2), 217-227.

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Detailed Immune Cell Profiling by Flow Cytometry

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Binding of the fusion protein was detected using PE-anti-human IgG Fc (clone HP6017,1 μg/ml). PD-L1 (clone 10 F.9G2, 1 μg/ml), IFNAR1 (clone MAR1-5A3, 1 μg/ml), CD45 (clone 30-F11, 0.5 μg/ml), CD80 (clone 16-10A1, 0.5 μg/ml), CD86 (clone GL1, 0.5 μg/ml) were from BioLegend or eBioscience. Cells suspended in FACS buffer (1% bovine serum albumin and 0.05% NaN3) were blocked with anti-CD16/32 (anti-FcγIII/ II receptor, clone 2.4G2) for 30 min and then stained with specific antibodies for 30 min on ice. Samples were analyzed on a FACS Calibur or Fortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (TreeStar).

Liang Y., Tang H., Guo J., Qiu X., Yang Z., Ren Z., Sun Z., Bian Y., Xu L., Xu H., Shen J., Han Y., Dong H., Peng H, & Fu Y.X. (2018). Targeting IFNα to tumor by anti-PD-L1 creates feedforward antitumor responses to overcome checkpoint blockade resistance. Nature Communications, 9, 4586.

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Multiparameter Flow Cytometric Analysis

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Mice were harvested at respective time points, single cell suspensions were prepared from spleens and tumors. Briefly, spleens were mashed and red blood cells were lysed by ACK lysis buffer. Tumors were pulverized and incubated in buffer containing 1mg/ml DNase and 1mg/ml collagenase at 45⁰C for 15 minutes. Spleens and tumor suspensions were passed through 60micron filters and cells were stained with the respective stain mixes. CD3(clone:145–2C11), CD4(clone: GK1.5), CD8(clone:53–6.7), CD69(clone:H1.2F3), CD11b(clone:M1/70), Ly6g(clone:IA8), Ly6C(clone:HK1.4) and PDL1(clone:10F.9G2) antibodies were purchased from Bio Legend (San Diego, CA). Cells were acquired through Fluorescence Activated Cell Sorter (FACS, BD biosciences, San Jose, CA, USA). Analysis was performed with Flow Jo software (Tree Star Inc., Ashland, OR, USA).

Varikuti S., Oghumu S., Elbaz M., Volpedo G., Ahirwar D.K., Alarcon P.C., Sperling R.H., Moretti E., Pioso M.S., Kimble J., Nasser M.W., Ganju R.K., Terrazas C, & Satoskar A.R. (2017). STAT1 gene deficient mice develop accelerated breast cancer growth and metastasis which is reduced by IL-17 blockade. Oncoimmunology, 6(11), e1361088.

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Multiparameter Flow Cytometry Analysis

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MOC cells were harvested and non-specific binding was blocked with anti-CD16/32 antibodies (Biolegend) prior to staining. Tissues were prepared into single-cell suspensions as previously described (14 (link)), followed by anti-CD16/32 antibody staining. Cell surface staining was performed using flourophore conjugated anti-mouse CD45.2 clone 104, CD3 clone 145-2C11, CD4 clone GK1.5, CD8 clone 53-6.7, NK1.1 clone PK136, CD31 clone 390, PD-L1 clone 10F.9G2, H2-K^b clone AF6-88.5, CD107a clone 1D4B, CD69 clone H1.2F3, PD-1 clone RMP1-30, CD11b clone M1/70, CD11c clone N418, CD80 clone 16-10A1, and CD86 clone GL-1 antibodies from Biolegend, 41BB clone 17B5 and OX40 clone OX-86 from eBioscience, and CD8 clone KT15 from MBL (Woburn, MA). H2Kb:KSPWFTTL (p15E_604–611) tetramer was purchased from MBL. Dead cells were excluded via 7AAD negativity. Isotype control antibodies and a “fluorescence minus one” method of antibody combination were used for specific staining validation. Data was acquired on a FACSCanto using FACSDiva software (BD Biosciences) and analyzed on FlowJo software vX10.0.7r2.

Moore E., Clavijo P.E., Davis R., Cash H., Van Waes C., Kim Y, & Allen C. (2016). Established T-cell inflamed tumors rejected after adaptive resistance was reversed by combination STING activation and PD-1–pathway blockade. Cancer immunology research, 4(12), 1061-1071.

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Multiparameter Flow Cytometry Staining

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For samples not sorted, cells were washed with PBS and stained in FACS buffer (2% FBS, 2 mM EDTA, and 0.001% NaN₃). All gating strategies are represented in Supplementary Fig. 9. Cells were first stained for H-2K^b/SIY-pentamer (PE; ProImmune) for 10 min at room temperature at a 1:20 dilution, followed by staining with remaining antibodies for 20 min on ice. Antibodies against the following molecules were used: CD3ε (clone 17A2, BioLegend, 100216), Thy1.2 (clone 30-H12, BioLegend, 105320), CD45.2 (clone 104, BioLegend, 109806), CD8α (clone 53-6.7, BioLegend, 100747), CD4 (clone RM4-5, BioLegend, 100547), PD-L1 (clone 10 F.9G2, BioLegend, 124312), CD19 (clone 6D5, BioLegend, 115545), I-A/I-E (clone M5/114.15.2, BioLegend, 107630), and H-2K^b (clone AF6-88.5, BioLegend, 742862). All antibodies were used at a 1:100 dilution. Fixable Viability Dye eFluor 506 or 780 (eBioscience) was used for live/dead discrimination and was used at a 1:200 dilution. All flow cytometric analysis was conducted on either an LSRFortessa or X20 (BD) and analyzed using FlowJo software (Tree Star).

Williams J.B., Li S., Higgs E.F., Cabanov A., Wang X., Huang H, & Gajewski T.F. (2020). Tumor heterogeneity and clonal cooperation influence the immune selection of IFN-γ-signaling mutant cancer cells. Nature Communications, 11, 602.

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Single-Cell Tumor Immune Profiling

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Tumor tissues were processed into single-cell suspensions by mincing, as well as chemical (Murine Tumor Dissociation Kit, Miltenyi Biotec) and mechanical (gentleMACS Dissociator) dissociation, per manufacturer recommendations. Suspensions were filtered through a 100 μm filter and washed with 1% BSA in PBS prior to blocking nonspecific staining with anti-CD16/32 (BioLegend) antibody. Cell surface staining was performed using fluorophore-conjugated anti–mouse CD45.2 (clone 104), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD31 (clone 390), PDGFR (clone APA5), PD-L1 (clone 10F.9G2), H2-K^b (clone AF6-88.5), CD107a (clone 1D4B), PD-1 (clone RMP1-30), CD11b (clone M1/70), Ly6G (clone 1A8), Ly6C (clone HK1.4), CTLA-4 (clone 9H10), CD39 (clone Duha59), CD11c (clone N418), F4/80 (clone BM8), and CD206 (clone C068C2) from BioLegend, and IL-12Rb2 (clone 305719) from R&D Systems. FoxP3⁺ Treg staining performed with the mouse Treg Staining Kit #1 (eBioscience) as per manufacturer’s protocol. Cell viability was assessed via staining with Sytox (Thermo Fisher Scientific) or Zombie (BioLegend) dyes. All analyses were performed on a BD Fortessa analyzer running FACSDiva software and interpreted using FlowJo V.X10.0.7r2.

Hong Y., Robbins Y., Yang X., Mydlarz W.K., Sowers A., Mitchell J.B., Gulley J.L., Schlom J., Gameiro S.R., Sievers C, & Allen C.T. (2022). Cure of syngeneic carcinomas with targeted IL-12 through obligate reprogramming of lymphoid and myeloid immunity. JCI Insight, 7(5), e157448.

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