Pe anti human cd8

Manufactured by BioLegend

Sourced in United States

PE anti-human CD8 is a fluorochrome-conjugated monoclonal antibody that binds to the CD8 receptor on the surface of human T cells. It is a core component used in flow cytometry and other immunoassays to identify and quantify CD8-expressing cells.

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10 protocols using pe anti human cd8

Comprehensive Flow Cytometry Analysis

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All samples were analyzed using a NovoCyteTM (ACEA Biosciences), LSR Fortessa, or C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies used included anti-MSLN-biotin (clone MB), Streptavidin-APC, anti-human CCR7-APC (clone 3D12), anti-human CD62L-PE (clone DREG-56), anti-human CD45RA-APC (clone HI100), anti-human CD45RO-PE (clone UCHL1), anti-human TIM3-PE (clone F38-2E2), anti-human LAG3-PerCP/Cy5.5 (clone 11C3C65), anti-human PD-1-APC (clone NAT105), anti-human CD27-PE (clone M-T271), anti-human CD28-APC (clone CD28.2), anti-human CD25-PE (clone BC96), anti-human CD69-APC (clone FN50), anti-human CD107a-APC (clone H4A3), anti-human CD3-APC (clone UCHT1), anti-human CD4-PerCP/Cy5.5 (clone OKT4), anti-human CD8-PE (clone OKT8), mouse IgG2a isotype control-APC (clone RMG2a-62), mouse IgG1kappa isotype control-PE, mouse IgG1kappa isotype control-PErCP/Cy5.5, and mouse IgG1kappa isotype control-APC (clone MOPC-21) (Biolegend, San Diego, CA, USA). All FACS-related staining procedures were performed on ice for 30 min, and cells were then washed with PBS containing 1% FBS before cytometry analysis. PB, spleen (SP), and tumor samples from mouse xenografts were treated with red blood cell lysis buffer (Biolegend), and the cells were stained with the corresponding antibodies.

Lv J., Zhao R., Wu D., Zheng D., Wu Z., Shi J., Wei X., Wu Q., Long Y., Lin S., Wang S., Wang Z., Li Y., Chen Y., He Q., Chen S., Yao H., Liu Z., Tang Z., Yao Y., Pei D., Liu P., Zhang X., Zhang Z., Cui S., Chen R, & Li P. (2019). Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer. Journal of Hematology & Oncology, 12, 18.

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Generation of Human T Cells from iPSCs

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A total of 30,000 cells per well of a 12-well plate were cultured on mitomycin C-treated OP9-hDLL1 in stromal culture medium with human cytokines as follow: 20 ng/mL FLT3L, 100 ng/mL SCF, 25 ng/mL IL-2, and 5 ng/mL IL-7 during the first 2 weeks and 10 ng/mL FLT3L, 25 ng/mL IL-2, and 5 ng/mL of IL-7 for the last 2 weeks. The cultures were transferred to fresh OP9-hDLL1 cells every 5 days after the first 10 days. For passaging, the cultures were treated with TrypLe (Thermo Fisher Scientific) and filtered through a 50-μm sterile Filcon (BD). Cells were analyzed after 1 month of culture using hCD45-PeCy5.5 (eBioscience), anti-human CD8-PE (BioLegend), and anti-human CD4-PeCy7 (eBioscience).

Garcia-Alegria E., Menegatti S., Fadlullah M.Z., Menendez P., Lacaud G, & Kouskoff V. (2018). Early Human Hemogenic Endothelium Generates Primitive and Definitive Hematopoiesis In Vitro. Stem Cell Reports, 11(5), 1061-1074.

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Multiparameter Flow Cytometry Analysis

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Primary antibodies used for this study includes Annexin V-FITC (Biolegend Cat# 640906), anti-PD-1-PE (BioLegend Cat# 621607), anti-human CD8-PE (BioLegend Cat# 344706), anti-mouse CD4-PE (BioLegend Cat# 100408), anti-mouse CD25-PE (BioLegend Cat# 102012), anti-mouse FOXP3-Alexa Fluor® 488 (BioLegend Cat# 126405), anti-mouse CD8-APC (BioLegend Cat# 100711), anti-mouse PD-1-PE (BioLegend Cat# 135205), IgG isotype control-PE (BioLegend Cat# 400907), anti-human CD4-APC (eBioscience Cat# RPA-T4). FoxP3 staining used FoxP3 buffer (BioLegend Cat#421403). Samples were analyzed using FACSCalibur and FlowJo software.

Wu G., Deng W., Chen H.Y., Cho H.J, & Kim J. (2024). Galectin 7 leads to a relative reduction in CD4+ T cells, mediated by PD-1. Scientific Reports, 14, 6625.

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Multicolor Flow Cytometry Immunophenotyping

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The following antibodies were used in this study: anti-human CD3 FITC (BioLegend), anti-human CD8 PE (BioLegend), anti-human CCR7 PE (BioLegend), anti-human CD45RA FITC (BioLegend), mouse anti-human CD69 Alexa Fluor 488 (BioLegend), and anti-human B220 APC (antigen-presenting cell; BioLegend). Chimeric FL-BCR expression was detected using anti-human IgG1 PE antibody (SouthernBiotech) or synthetic biotinylated cyclopeptides (GeneCust) and streptavidin conjugated with FITC or PE (Thermo Fisher Scientific). The CAR molecules were detected using goat cross-absorbed anti-human IgG antibody conjugated with DyLight 650 (Thermo Fisher Scientific). The CD19-CAR (FMC63 clone) molecules were detected using biotinylated protein L (Thermo Fisher Scientific) and streptavidin conjugated with FITC (Thermo Fisher Scientific).

Stepanov A.V., Markov O.V., Chernikov I.V., Gladkikh D.V., Zhang H., Jones T., Sen’kova A.V., Chernolovskaya E.L., Zenkova M.A., Kalinin R.S., Rubtsova M.P., Meleshko A.N., Genkin D.D., Belogurov AA J.r., Xie J., Gabibov A.G, & Lerner R.A. (2018). Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma. Science Advances, 4(11), eaau4580.

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Evaluation of HVEM on T Lymphocytes

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Whole blood was obtained in heparin containing tubes. A portion of each sample was separated to collect serum which was then stored at −80°C for later cytokine analysis. The remaining whole blood was layered with Ficoll Histopaque −1077 and centrifuged. The layer containing leukocytes was aspirated and washed with phosphate buffered saline then centrifuged. Cells were stained using monoclonal antibodies for CD3 (BD Pharmingen, FITC Mouse Anti-Human CD3, Cat#555332; Beckman Coulter, APC Anti-Human CD3, PN#IM2467U), CD4 (Biolegend, PE-anti-human CD4, Cat# 318806) or CD8 (Biolegend, PE-anti-human CD8, Cat#344706) to identify lymphocytes, as well as for HVEM (Biolegend, PE-anti-human CD270, Clone 122, Cat#318806). Cellular populations were analyzed using flow cytometry. Initially, distinct forward and side scatter patterns were used to identify lymphocytes. Gating based on isotype controls was then used to identify HVEM⁺CD3⁺ lymphocytes. Flow cytometry data analysis was completed using FlowJo software.
For cytokine analysis we chose to examine patient plasma IL-6, IL-10, IL-2 and TNF-α levels using a Cytometric Bead Array (CBA) (BD Biosciences) according to the manufacture’s specifications and as we have previously described^{23 (link)}.

Wakeley M.E., Shubin N.J., Monaghan S.F., Gray C.C., Ayala A, & Heffernan D.S. (2019). Herpes Virus Entry Mediator(HVEM): A novel potential mediator of trauma induced immunosuppression. The Journal of surgical research, 245, 610-618.

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CA9 DATEs Modulate T Cell Responses in ccRCC and GBM

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In ccRCC model, RCC243 and RCC243 CA9-KO cells were plated at 200,000 cells/well in 6 well plates the night prior to treatment. Human CD3⁺ T cells at an E:T ratio of 5:1 were added to the wells along with (1 nM) or without CA9 DATEs and incubated at 37°C in 5% CO₂ for 48 hours. The T cells were collected and stained for BV785 anti-human CD3 (BioLegend, Cat#317330), BV605 anti-human CD4 (BioLegend, Cat#317438), PE-anti-human CD8 (BioLegend, Cat#300908), and PE-CF594-anti-CD25 (BD Biosciences, Cat#562403) antibodies. Supernatants were collected and stored at -80°C for cytokine release analysis by enzyme-linked immunosorbent assay (ELISA).
In GBM models, GBM cells and T cells were co-incubated at a 1:1 ratio for 24 hours with (1μg = 13 nM) or without CA9 DATEs. The CD3⁺ (BD Pharmingen, Cat#563423) T cells and subpopulation of T cells including CD4⁺ (BD Pharmingen, Cat#555347) and CD8⁺ T (BD Horizon, Cat#562428) cells were analyzed for activation markers CD25 (Miltenyi Biotech, Cat#130-113-283) and CD69 (BD Pharmingen, Cat#555533) by flow cytometry. Supernatants were collected and stored at -80°C for cytokine release analysis by enzyme-linked immunosorbent assay (ELISA).

Tatari N., Zhang X., Chafe S.C., McKenna D., Lawson K.A., Subapanditha M., Shaikh M.V., Seyfrid M., Savage N., Venugopal C., Moffat J, & Singh S.K. (2022). Dual Antigen T Cell Engagers Targeting CA9 as an Effective Immunotherapeutic Modality for Targeting CA9 in Solid Tumors. Frontiers in Immunology, 13, 905768.

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Multiparameter Flow Cytometry of PBMCs

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PBMCs were stained with PerCP-Cyanine 5.5 anti-human CD4 (clone # OKT4, Biolegend, San Diego, CA), PE anti-human CD8 (clone # SK1, Biolegend), FITC anti-human TCR V alpha 2 (clone # F1, Invitrogen, Waltham, MA) and FITC anti-human TCR V beta 3.1 (clone # 8F10, Invitrogen). Cells were acquired on a CytoFLEX LX flow cytometer (Beckman Coulter, Pasadena, CA) and analyzed using FlowJo 2 software (version 10, TreeStar, Ashland, OR). Fluorescence compensation was adjusted based on fluorescence-minus-one method.

Wang N., Vuerich M., Kalbasi A., Graham J.J., Csizmadia E., Manickas-Hill Z.J., Woolley A., David C., Miller E.M., Gorman K., Hecht J.L., Shaefi S., Robson S.C, & Longhi M.S. (2021). Limited TCR repertoire and ENTPD1 dysregulation mark late-stage COVID-19. iScience, 24(10), 103205.

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Flow Cytometry Analysis of CD Markers

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According to the manufacturer protocol, cells and CD19 CAR lentivirus infected cells were washed and stained with PE anti-human CD5 (BioLegend), APC anti-human CD7 (BioLegend), PE anti-human CD8 (BioLegend), FITC and APC anti-human CD3 (BioLegend), FITC anti-human CD4 (BioLegend), PE anti-human CD34 (Elabscience), and/or APC and FITC anti-human EGFR (BioLegend) antibodies at 4°C in the dark for 30 min. They were analyzed using a BD Influx flow cytometer (Beckman CytoFLEX, California, United States). Maintaining the standard manufacturer protocol, the cell cycle in Raji and A549 cells was also analyzed using flow cytometry. (Jian et al., 2023 (link)).

Chen Y., Huang P., Niu M., Tian C., Zhang T, & Peng Z. (2023). Regeneration of T cells from human-induced pluripotent stem cells for CAR-T cell medicated immunotherapy. Frontiers in Bioengineering and Biotechnology, 11, 1159507.

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Cell Immunophenotyping by Flow Cytometry

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One million cells were suspended in 100 μL PBS and incubated with antibodies for 60 min on ice in the dark. The antibodies used were PerCP anti-human CD3, FITC anti-human CD4, PE anti-human CD8, PE anti-mouse CD4, and FITC anti-mouse CD8a (BioLegend, San Diego, CA, USA). After washing with PBS, the cells were resuspended in 500 μL PBS and measured using a CANTO II system (Beckton-Dickinson, Franklin Lakes, NJ, USA). All data were analyzed using FlowJo v10.7.1 software (Ashland, OR, USA).

Sasaki K., Takano S., Tomizawa S., Miyahara Y., Furukawa K., Takayashiki T., Kuboki S., Takada M, & Ohtsuka M. (2021). C4b-binding protein α-chain enhances antitumor immunity by facilitating the accumulation of tumor-infiltrating lymphocytes in the tumor microenvironment in pancreatic cancer. Journal of Experimental & Clinical Cancer Research : CR, 40, 212.

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Phenotypic Profiling of PBMCs

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Lymphoprep® (Axis-Shield PoC AS, Oslo, Norway) was used to separate the whole blood into peripheral blood mononuclear cells (PBMCs). PBMCs were stained with antibodies, including FITC anti-human TCRαβ (BD Biosciences Pharmingen, San Diego, CA, USA), PE/Cy7 anti-human CD3 (Biolegend, San Diego, CA, USA), Paci cBlue anti-human CD3 (Biolegend), PE anti-human CD4 (Biolegend), PE/Cy7 antihuman CD4 (Biolegend), PE anti-human CD8 (Biolegend), APC anti-human CD8 (Biolegend), PE antihuman CD45RO (BD Biosciences Pharmingen), PE anti-human CCR6 (BD Biosciences Pharmingen), APC anti-human CCR4 (Biolegend), and APC anti-human CXCR3 (Biolegend), in a dark place at 4℃ for 30 min.
The PBMCs were xed in intracellular staining of RORγt and intracellularly stained using Fixation/Permeabilization Concentrate (eBioscience, San Diego, CA, USA), Fixation/Permeabilization Diluent (eBioscience), and Permeabilization buffer (eBioscience) with PE anti-human RORγt antibody (BD Biosciences Pharmingen) in a dark place at 4℃ for 45 min. The Cell Analyzer EC800 (Sony Biotechnology, Tokyo, Japan) was used for ow cytometric analysis. The data analysis was performed using FlowJo 7.6.5 software (TreeStar Inc, Ashland, OR, United States).

Kurita D., Shiba N., Ohya T., Murase A., Shimosato Y., Yoshitomi M., Hattori S., Sasaki K., Nishimura K., Tsujimoto S.I., Takeuchi M., Tanoshima R., Kanegane H., Kitagawa N, & Ito S. (2023). Severe RAS-Associated Lymphoproliferative Disease Case with Increasing αβ Double-Negative T Cells with Atypical Features. Journal of clinical immunology, 43(8).

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