Anti pd 1 clone 29f 1a12

Manufactured by BioLegend

Sourced in United Kingdom, United States

Anti-PD-1 (clone 29F.1A12) is a monoclonal antibody that recognizes the programmed cell death-1 (PD-1) receptor. PD-1 is an inhibitory receptor expressed on activated T cells, B cells, and myeloid cells, and plays a role in regulating immune responses.

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Anti-PD-1 (51 citations) PD-1 (29F.1A12; (19 citations) Anti-PD-1 (29F.1A12, (6 citations) Anti-PD-1-BV785 (clone 29F.1A12) (3 citations) Anti‐mouse CD279 (PD‐1) antibody (clone 29F.1A12) (2 citations) Anti-PD-1 APC (clone 29F-1A12), (2 citations)

11 protocols using anti pd 1 clone 29f 1a12

Evaluating Immune Checkpoint Expression

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Mouse spleen cells were washed with PBS containing 1% BSA (staining buffer), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies. Antibodies used were anti-PD-1 (clone 29F.1A12, Biolegend, San Diego, CA), anti-TIGIT (clone 1G9, Biolegend), anti-GITR (clone yGITR765, Biolegend), anti-CD25 (clone PC61, Biolegend), anti-PD-L1 (clone 10F.9G2, Biolegend), anti-CTLA4 (clone UC10-4B9, Biolegend), anti-2B4 (clone M2B4(B6)458.1, Biolegend), anti-VISTA (clone MIH63, Biolegend), anti-LAG3 (clone C9B7W, Biolegend), anti-TIM3 (clone RMT3-23, Biolegend), and anti-CD4 (clone RM4-5, Biolegend).
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), anti-CD25 (clone BC96, Biolegend), anti-FoxP3 (clone 236A/E7, Biolegend), anti-PD-1 (clone EH12.2H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.

Muhammad F., Wang D., McDonald T., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2020). TIGIT+ A2Ar-Dependent Anti-Uveitic Treg Cells are a Novel Subset of Tregs Associated with Resolution of Autoimmune Uveitis. Journal of autoimmunity, 111, 102441.

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Comprehensive Splenic T Cell Analysis

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Spleens were harvested after mice were sacrificed 48 hours after CLP, and then were processed to single-cell suspensions though a 70 μm filter. Splenocytes were rinsed with 10 mL cold PBS, and 200 μL from each spleen was put into a 96-well plate for staining. Anti-CD3 (BD, clone 500A2), anti-CD4 (clone RM4-5, BioLegend), anti-CD8 (clone MCD0830, Invitrogen), anti-CD44 ( clone IM7, BioLegend), anti-CD226 (clone 10E5, BioLegend), anti-PD-1 (clone 29F.1A12, BioLegend), anti-2B4 (clone eBio244F4, Thermo Fisher Scientific), and anti-TIM-3 (clone RMT3-23, BioLegend) were used for surface staining to determine T cell phenotype. For the detection of cell apoptosis, splenocytes were stained with a FITC Annexin V apoptosis detection kit with 7-AAD (BioLegend). Tregs were identified via intracellular staining for Foxp3-APC (clone FJK-16S, eBioscience). Splenocytes were surface stained for anti-CD62L (MEL-14), anti-CD69 (H1.2F3), and then permeabilized using a Foxp3 kit (BD Biosciences) and stained with anti-Foxp3, anti-CTLA-4 (UC10-489), and anti-Helios (22F6, all Abs from BioLegend). AccuCheck Counting Beads (Thermo Fisher Scientific) were added after staining to calculate the absolute number of cells per spleen.

Sun Y., Anyalebechi J.C., Sun H., Yumoto T., Xue M., Liu D., Liang Z., Coopersmith C.M, & Ford M.L. (2021). Anti-TIGIT differentially affects sepsis survival in immunologically experienced versus previously naive hosts. JCI Insight, 6(5), e141245.

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Flow Cytometry Analysis of Mouse and Human Immune Cells

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Staining of mouse cells for flow cytometry analysis was done after cells were collected and washed with PBS with 1% BSA (staining buffer), and then Fc-blocked with mouse IgG. Conjugated antibodies used were anti-PD-1 (clone 29F.1A12, Biolegend, San Diego, CA), anti-TIGIT (clone 1G9, Biolegend), anti-CD4 (clone RM4-5, Biolegend), anti-CCR6 (clone 29-2L17), anti-CCR7 (clone 4B12), anti-CXCR6 (clone SA051D1). Mouse spleen cells were washed with staining buffer (PBS + 1% BSA), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies.
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236A/E7, Biolegend), human PD-1 (clone EH12.2H7, Biolegend), CCR6 (clone GO34E3, Biolegend), anti-CCR7 (clone GO43H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences, Franklin Lakes, NJ) or in the Ocular Immunobiology P30 Core Module at Dean McGee Eye Institute on a Cytek Aurora (Cytek, Fremont, CA). Spectral unmixing of full spectrum flow cytometry data was done with SpectroFlo software (Cytek) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).

Peters K., McDonald T., Muhammad F., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2023). A2Ar-dependent PD-1+ and TIGIT+ Treg cells have distinct homing requirements to suppress autoimmune uveitis in mice. Mucosal immunology, 16(4), 422-431.

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Multiparameter Flow Cytometry Analysis

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Single cell suspensions of spleens were blocked with anti-mouse CD16/32 (BioLegend) and surface stained with the indicated markers, and evaluated by flow cytometric analysis as described^{18 (link),29 (link),35 (link)–37 (link)}. The following Abs were used; anti-CD45 (clone 30-F11, Invitrogen), anti-PD-1 (clone 29F.1A12, BioLegend), anti-CX3CR1 (clone SA011F11, BioLegend), anti-KLRG1 (clone 2F1/KLRG1, BioLegend), anti-CD4 (clone GK1.5, BioLegend), anti-CD8α (clone 53-6.7, BD Biosciences), and anti-CD3 (clone 145-2C11, BioLegend). LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Thermo Fisher Scientific)-stained cells were excluded from the analysis. Samples were acquired by Fortessa (BD Biosciences) cytometers, and analyzed with FlowJo software (Treestar).

Yokoi T., Oba T., Kajihara R., Abrams S.I, & Ito F. (2021). Local, multimodal intralesional therapy renders distant brain metastases susceptible to PD-L1 blockade in a preclinical model of triple-negative breast cancer. Scientific Reports, 11, 21992.

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Comprehensive Immunohistochemical Analysis of Lung Samples

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Lung samples were formalin- or Zn- fixed followed by paraffin embedding and immunostaining of 5 µm sections as previously described (21 (link)). H&E staining were preformed as reported before (23 (link)). Sections were stained with the following antibodies: anti-MCP-1 (BD Biosciences), anti-CD86 (BD Biosciences), anti-CD3 (Epitomics), anti-PD-1 (clone 29F.1A12, Biolegend), anti-PDL-1 (clone 10F.9G2, Biolegend), anti-granzyme B (Abcam) and anti-VEGF (Thermo Scientific).

Seth P., Csizmadia E., Hedblom A., Vuerich M., Xie H., Li M., Longhi M.S, & Wegiel B. (2017). Deletion of lactate dehydrogenase-A in myeloid cells triggers antitumor immunity. Cancer research, 77(13), 3632-3643.

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Investigating Immune Checkpoint Proteins

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Antibody sources were as follows: anti–PD-1 (clone 29F.1A12; BioLegend, San Diego, Calif), anti–PD-L1 (clone 10F.9G2; BioLegend), anti-NKG2D (clone 1D11; BioLegend), anti-CD16 (clone 3G8; Bio-Legend), anti–LFA-1 (clone H155–78; BioLegend), anti-perforin (clone δG9; Thermo Fisher), anti-ILK (clone EPR1592; Abcam, Cambridge, United Kingdom), and anti-actin (clone C4; Santa Cruz Biotechnology).

Huang Y., Chen Z., Jang J.H., Baig M.S., Bertolet G., Schroeder C., Huang S., Hu Q., Zhao Y., Lewis D.E., Qin L., Zhu M.X, & Liu D. (2018). PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse. The Journal of allergy and clinical immunology, 142(4), 1311-1321.e8.

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Detailed Immune Cell Profiling

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Phenotype analysis was performed with staining performed at 4 °C for 20 min with the following antibodies: anti‐mouse CD45 (clone 30‐F11; Biolegend, San Diego, CA, USA), anti‐mouse F4/80 (clone BM8; Biolegend), anti‐mouse CD11c (clone N418; Biolegend), anti‐mouse Ly6C (clone HK1.4; Thermo Fisher Scientific); anti‐CD11b (clone M1/70; Biolegend, San Diego, CA, USA), anti‐CD206 (clone C068C2; Biolegend), anti‐Ly6G (clone 1A8; Biolegend), anti‐NK1.1 (clone PK136; Biolegend), anti‐CD314 (clone C7; Biolegend), anti‐CD4 (clone GK1.5; BD Pharmigen, San Jose, CA, USA), anti CD8 (clone 53‐6.7; BD Pharmigen); antiPD‐1 (clone 29F.1A12; Biolegend), anti‐PD‐L1 (clone 10F.9G2; Biolegend). Cells were detected using the Cyan ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed with the Summit 4.3 software (Beckman Coulter). Quadrants were set based on isotype control antibody, and cells were gated among total DAPI⁻ cells.

Comunanza V., Gigliotti C., Lamba S., Doronzo G., Vallariello E., Martin V., Isella C., Medico E., Bardelli A., Sangiolo D., Di Nicolantonio F, & Bussolino F. (2023). Dual VEGFA/BRAF targeting boosts PD‐1 blockade in melanoma through GM‐CSF‐mediated infiltration of M1 macrophages. Molecular Oncology, 17(8), 1474-1491.

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Flow Cytometry Analysis of Mouse and Human Immune Cells

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Multiparameter Immune Cell Profiling

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Mouse spleen cells were washed with PBS with 1% BSA (staining buffer), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies. Antibodies used were anti-CD11b (clone M1/70, Biolegend, San Diego, CA), anti-Ly-6C (clone HK1.4, Biolegend), anti-Ly-6G (clone 1A8, Biolegend), anti-F4/80 (clone BM8, eBiosciences, San Diego, CA), anti-MHCII (clone M5/114.15.2, Biolegend), anti-CD4 (clone RM4-5, Biolegend), anti-PD-1 (clone 29F.1A12, Biolegend), anti-Foxp3 (clone FJK-16s, eBiosciences), anti-Tbet (clone 4B10, Biolegend), and anti-RORγt (clone AFKJS-9, Biolegend). Prior to anti-Foxp3, anti-Tbet, and anti-RORγt staining, the cells were fixed and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences) or the DMEI Ocular Immunobiology Core on a 4-laser Aurora (Cytek Biosciences, Fremont, CA). When the Aurora was used, unmixing was done using SpectroFlo Software (Cytek) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).

McDonald T., Muhammad F., Peters K, & Lee D.J. (2021). Combined Deficiency of the Melanocortin 5 Receptor and Adenosine 2A Receptor Unexpectedly Provides Resistance to Autoimmune Disease in a CD8+ T Cell-Dependent Manner. Frontiers in Immunology, 12, 742154.

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Sorting Reactivated Spleen T Cells

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Reactivated spleen T cells from post-EAU mice were stained as described above. Antibodies used to stain T cells were anti-CD4 (clone GK1.5, Biolegend, San Diego, CA), anti-CD25 (clone PC61, Biolegend), and anti-PD-1 (clone 29 F.1A12, Biolegend). Gating was determined using single stained samples. Stained cells were sorted in the Oklahoma Medical Research Foundation Flow Cytometry Core on a FACSAria III (BD Biosciences). Cells were sorted into tubes containing 10% FBS and found to be ≥98% pure.

Muhammad F., Wang D., Montieth A., Lee S., Preble J., Foster C.S., Larson T.A., Ding K., Dvorak J.D, & Lee D.J. (2019). PD-1+ melanocortin receptor dependent-Treg cells prevent autoimmune disease. Scientific Reports, 9, 16941.

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