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Anti cd80 clone 16 10a1

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Anti-CD80 (clone 16-10A1) is a monoclonal antibody that specifically binds to the CD80 surface protein, also known as B7-1. CD80 is a co-stimulatory molecule expressed on the surface of antigen-presenting cells and plays a crucial role in T cell activation and regulation of the immune response.

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Lab products found in correlation

Anti cd45 clone 30 f11, by Thermo Fisher Scientific (5 mentions) Anti cd11c clone n418, by BioLegend (4 mentions) Anti ifn γ clone xmg1, by BioLegend (3 mentions) Streptavidin pe cy7, by Thermo Fisher Scientific (3 mentions) Anti epcam1 clone g8, by Thermo Fisher Scientific (3 mentions) Anti ly51 clone 6c3, by BioLegend (3 mentions) Cd104 clone 346 11a, by BioLegend (3 mentions) Facsaria fusion 1 cell sorter, by BD (3 mentions) Biotinylated uea 1, by Vector Laboratories (3 mentions) Anti mhc 2 clone m5 114, by Thermo Fisher Scientific (3 mentions) Lsrfortessa, by BD (3 mentions) Dnase 1, by Roche (3 mentions) Collagenase dispase, by Roche (3 mentions) Anti cd86 clone gl 1, by BioLegend (2 mentions) Anti cd4 clone rm4 5, by BioLegend (2 mentions) Facsaria 3 platform, by BD (2 mentions) Anti fcr clone 2.4 g2, by BD (2 mentions) Anti cd86 clone gl1, by Thermo Fisher Scientific (2 mentions) Anti cd8 clone 53 6, by BioLegend (2 mentions) Clone 25d1.16, by BioLegend (2 mentions)

Spelling variants of this product

Anti-CD80 (37 citations) CD80 (16-10A1), (12 citations) CD80 (clone 16-10A1, (6 citations) Anti-CD80 (16-10A1, (4 citations) PE anti-mouse CD80 clone 16-10A1 (3 citations) Clone 16-10A1 (2 citations) Anti-mouse CD80 (clone 16-10A1), (2 citations)

9 protocols using anti cd80 clone 16 10a1

1

Phenotypic Characterization of Cells

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Cells were stained with live/dead Aqua (Thermofisher L34965) prior to staining with anti-MHCII (clone M5.114.15.2, Biolegend), anti-CD80 (clone 16-10A1, Biolegend) and anti- CD86 (clone GL-1, Biolegend) (Fig 4) on ice for 30 minutes. Cells were washed and fixed in 2% para-formaldehyde (VWR 76221–378) prior to acquisition on an LSRII instrument. For sorting experiments cells were stained with live/dead Aqua (Thermofisher L34965) prior to staining with anti-HA (clone 16B12, Biolegend) and sorted using a MoFLo Astrios or FACS Jazz instrument (Children’s Hospital of Philadelphia Research Institute Flow Cytometry Core Facility).
Forsyth K.S., Roy N.H., Peauroi E., DeHaven B.C., Wold E.D., Hersperger A.R., Burkhardt J.K, & Eisenlohr L.C. (2020). Ectromelia-encoded virulence factor C15 specifically inhibits antigen presentation to CD4+ T cells post peptide loading. PLoS Pathogens, 16(8), e1008685.
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2

Tumor-Infiltrating Immune Cell Analysis

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Tumors were harvested and dissociated into single-cell suspensions. Then, cells were blocked with anti‐FcR (clone 2.4 G2, BD Pharmingen) and labeled with indicated surface markers for 30 min at 4°C. For IFN-γ staining, single-cells were cultured in the presence of a cell activation cocktail (with Brefeldin A) (Biolegend) for 5 h. Cells were permeabilized and stained with intracellular antibodies for 30 min at 4°C as instructed by the manufacturer. Dead cells were excluded using LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). The antibodies used in the flow cytometry analysis were: anti-CD45 (clone 30-F11, Invitrogen), anti-CD3 (clone 145–2C11, Biolegend), anti CD4 (clone RM4-5, Biolegend), anti-CD8 (clone 53–6.7, Biolegend), anti-IFN-γ (clone XMG1.2, Biolegend), anti-CD11C (clone N418, Biolegend), anti-CD80 (clone 16–10A1, Biolegend), and anti-CD86 (clone GL1, Invitrogen). Because the specific reaction with ovalbumin-derived peptide SIINFEKL bound to H-2 Kb of MHC class I, anti–H-2kb bound to SIINFEKL (clone 25-D1.16, Biolegend) was used to recognize the tumor specific immune cells. Flow cytometry was performed on the FACS Aria III platform (BD Biosciences, San Jose, CA, USA) and results analyzed using FlowJo software version 10.4 (TreeStar).
Li Z., Zhang Y., Hong W., Wang B., Chen Y., Yang P., Zhou J., Fan J., Zeng Z, & Du S. (2022). Gut microbiota modulate radiotherapy-associated antitumor immune responses against hepatocellular carcinoma Via STING signaling. Gut Microbes, 14(1), 2119055.
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3

Tumor-specific T Cell Identification

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The tumors were harvested and dissociated into single‐cell suspensions. The cells were then blocked with anti‐FcR (clone 2.4G2, BD Pharmingen) and labeled with indicated surface markers for 30 min at 4 °C. For IFN‐γ staining, single‐cells were cultured in the presence of a cell activation cocktail (with Brefeldin A; BioLegend) for 5 h. Cells were permeabilized and stained with intracellular antibodies for 30 min at 4 °C as instructed by the manufacturer. Dead cells were excluded using a LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). The antibodies used in the flow cytometry analysis were anti‐CD45 (clone 30‐F11, Invitrogen), anti‐CD3 (clone 145‐2C11, BioLegend), anti‐CD4 (clone RM4‐5, BioLegend), anti‐CD8 (clone 53–6.7, BioLegend), anti‐IFN‐γ (clone XMG1.2, BioLegend), anti‐CD11c (clone N418, BioLegend), anti‐CD80 (clone 16‐10A1, BioLegend), and anti‐CD86 (clone GL1, Invitrogen). Because the specific reaction with the ovalbumin‐derived peptide SIINFEKL bound to H‐2Kb of MHC class I, anti–H‐2 kb bound to SIINFEKL (clone 25‐D1.16, BioLegend) was used to recognize the tumor‐specific immune cells. Flow cytometry was performed on a FACS Aria III platform (BD Biosciences, San Jose, CA, USA), and the results were analyzed using FlowJo software version 10.4 (TreeStar).
Hong W., Zhang Y., Wang S., Li Z., Zheng D., Hsu S., Zhou J., Fan J., Chen Z., Xia X., Zeng Z., Gao Q., Yu M, & Du S. (2024). RECQL4 Inhibits Radiation‐Induced Tumor Immune Awakening via Suppressing the cGAS‐STING Pathway in Hepatocellular Carcinoma. Advanced Science, 11(16), 2308009.
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4

Phenotypic and Functional Analysis of DCs

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For phenotypic analysis, the surface molecules of DCs were stained with monoclonal antibody-conjugated fluorescent agent. Cells were incubated with the following antibodies at 4 °C for 20 min: anti-CD11c (clone N418, allophycocyanin (APC) or phycoerythrin (PE)), anti-CD80 (clone 16-10A1, PE), anti-CD86 (clone PO3, fluorescein-5-isothiocyanate (FITC)), and anti-MHC II (clone 2G9, PE) (BioLegend or eBioscience, San Diego, CA, USA). To examine T cell subpopulations, cells were stained with APC-conjugated anti-CD4 (clone RM4-5), and then cells were fixed and permeabilized using an Intracellular Staining Kit (BD Biosciences, San Diego, CA, USA or Invitrogen, Waltham, MA, USA). For intracellular staining, anti-IFN-γ (clone XMG1.2, PE), anti-IL-4 (clone 11B11, Alexa Fluor 488), or anti-IL-17A (clone TC11-18H10.1, PE) (all from BD Bioscience) plus anti-Foxp3 (clone FJK-16s; Invitrogen, PE) antibodies were used. FlowJo v.10.6.2 software (FlowJo, Ashland, OR, USA) was used to analyze the data.
Oh J.S., Hwang S.U., Noh K.E., Lee J.H., Choi S.Y., Nam J.H., Song M.S., Jung N.C., Song J.Y., Seo H.G., Na Y, & Lim D.S. (2022). Synthetic TGF-β Signaling Agonist-Treated Dendritic Cells Induce Tolerogenicity and Antirheumatic Effects. Current Issues in Molecular Biology, 44(9), 3809-3821.
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5

Thymic Epithelial Cell Isolation and Analysis

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For thymic epithelial cell analysis, single cell suspensions were generated by digesting thymic lobes with collagenase dispase (2.5mg/ml, Roche) and DNase 1 (40mg/ml Roche). CD45- cells were enriched by the depletion of CD45+ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following antibodies were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (Biolegend), anti-MHCII clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (Biolegend), CD104 clone 346-11A (Biolegend), and anti-MHCI 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analysed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using Flowjo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the antibodies above, and isolated using a FACS Aria Fusion 1 cell sorter (Becton Dickinson).
The sorting strategy for the different TEC subsets were as follows, Cxcl12DsRed+ cTEC: CD45-EpCAM1+UEA1-Ly51+CXCL12DsRed+; CXCL12DsRed- cTEC: CD45-EpCAM1+UEA1-Ly51+CXCL12DsRed-; mTEClo: CD45-EpCAM1+UEA1+Ly51-CD80-MHCII-; mTEChi: CD45-EpCAM1+UEA-1-Ly51+CD80+MHCII+; CD104+ mTEClo, CD45-EpCAM1+UEA1+Ly51-CD80-MHCII-CD104+; CD104- mTEClo, CD45-EpCAM1+UEA1+Ly51-CD80-MHCII-CD104-.
White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.
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6

Proliferation Assay of Regulatory T Cells

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Proliferation Assays. CD4+CD25high (all Foxp3+) cells isolated from the secondary lymphoid organs were magnetically sorted from the spleen of Foxp3-GFP-KI C57BL/6J mice. They were loaded with 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies) and cultured (5× 104 cells per well) in RPMI medium 1640 supplemented with 5% (vol/vol) FCS (Life Technologies), 1% antibiotics, and 5 × 10−5 M β-mercaptoethanol. Cells were plated in 96-well round-bottomed culture plates, either alone or with sorted MPP at 1:1 T:MPP cell ratios, and stimulated with 2.5 μg/ml of anti-CD3 mAb (clone 145–2C11) and 5 μg/ml of anti-CD28 mAb (clone 37.51, eBioscience) for 4 days. Inhibitors were added at 5 to 20 µg/ml: anti-CD137L (clone TKS-1, Biolegend), anti-CD80 (clone 16-10A1, Biolegend), anti-GITRL (clone 5F1, Biolegend), anti-CD86 (clone GL-1, BD Biosciences), anti-OX40 (polyclonal goat IgG, R&D Systems), anti-TGFβ (clone 2G7, grown in our laboratory), anti-IL10 (clone JES052A5, R&D).
Korniotis S., D’Aveni M., Hergalant S., Letscher H., Tejerina E., Gastineau P., Agbogan V.A., Gras C., Fouquet G., Rossignol J., Chèvre J.C., Cagnard N., Rubio M.T., Hermine O, & Zavala F. (2020). Mobilized Multipotent Hematopoietic Progenitors Stabilize and Expand Regulatory T Cells to Protect Against Autoimmune Encephalomyelitis. Frontiers in Immunology, 11, 607175.
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7

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

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For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45− cells were enriched by the depletion of CD45+ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12DsRed+ cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed+; CXCL12DsRed− cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed−; mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−; mTEChi, CD45−EpCAM1+UEA+Ly51−CD80+MHC II+; CD104+ mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104+; and CD104− mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104−.
White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.
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8

Flow Cytometric Analysis of Immune Cells

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The primary antibodies used for flow cytometry were as follows: anti‐F4/80 (clone BM8, BioLegend), anti‐CD11c (clone N418, BioLegend), anti‐MHC II (clone 10‐3.6, BioLegend), anti‐PDCA1 (clone 927, BioLegend), anti‐Gr1 (clone RB6‐8C5, BioLegend), anti‐CD4 (clone GK1.5, BioLegend), anti‐CD8a (clone 53‐6.7, BioLegend), anti‐IFN‐γ (clone XMG1.2, BioLegend), anti‐CD44 (clone IM7, BioLegend), anti‐CD80 (clone 16‐10A1, BioLegend), anti‐CD62L (clone MEL‐14, BioLegend), anti‐CD11b (clone M1/70, BioLegend), anti‐CD86 (clone GL‐1, BioLegend), anti‐I‐A/I‐E (clone M5/114.15.2, BioLegend), DC Marker (clone 33D1, BioLegend), anti‐Siglec‐H (clone 551, BioLegend), anti‐CD3ε (clone 145‐2C11, BioLegend), anti‐CD19 (clone 6D5, BioLegend), anti‐Ly‐6G (clone 1A8, BioLegend) and anti‐CD335 (clone 29A1.4, BioLegend). Mononuclear cells were isolated from the spleen and draining lymph nodes. A total of 1 × 106 cells were incubated with 1·5 mg mL−1 antibody for 30 min at 4°C. For intracellular staining, cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience) for 30 min at 4°C. The cells were then washed and stained for 30 min at 4°C with antibodies diluted with permeabilization buffer. The cell suspensions were analysed on an LSRII flow cytometer (BD Biosciences), and the data were analysed using FlowJo.
Geng G., Xu C., Peng N., Li Y., Liu J., Wu J., Liang J., Zhu Y, & Shi L. (2021). PTBP1 is necessary for dendritic cells to regulate T‐cell homeostasis and antitumour immunity. Immunology, 163(1), 74-85.
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9

Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells

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For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45− cells were enriched by the depletion of CD45+ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12DsRed+ cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed+; CXCL12DsRed− cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed−; mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−; mTEChi, CD45−EpCAM1+UEA+Ly51−CD80+MHC II+; CD104+ mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104+; and CD104− mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104−.
White A.J., Parnell S.M., Handel A., Maio S., Bacon A., Cosway E.J., Lucas B., James K.D., Cowan J.E., Jenkinson W.E., Hollander G.A, & Anderson G. (2023). Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk. The Journal of Immunology Author Choice, 210(1), 40-49.
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