Anti cd8 bv510

Manufactured by BioLegend

Anti-CD8-BV510 is a monoclonal antibody that binds to the CD8 antigen. CD8 is a glycoprotein that is expressed on the surface of cytotoxic T cells and a subset of natural killer cells. The antibody is conjugated to the BV510 fluorophore, which can be used for flow cytometry applications.

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10 protocols using anti cd8 bv510

Apoptosis Detection in T Cells

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Annexin V/PI and Annexin V/7-AAD Staining Kit (Beyotime) were used to detect cell apoptosis. Splenic cells or peripheral blood in mice were stained with anti-CD3 (APC), anti-CD4 (PE-CY7) and anti-CD8 (BV510) antibodies (Biolegend), as well as Annexin V-PE and 7-AAD. Human peripheral blood was stained with anti-human CD3 (FITC), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies as well as Annexin V-PE and 7-AAD. Purified CD3⁺ T cells obtained from MRL/lpr mice were incubated with DMSO or MLN4924 (0.1 and 0.5 μM) for 12 h and then stained with the indicated antibodies: anti-CD3 (APC), anti-CD4 (PE-CY7) and anti-CD8 (BV510) (Biolegend). Peripheral Blood Mononuclear Cell (PBMC) isolated from patients were treated with 0.5 μM MLN4924 for 6 h and then stained with anti-human CD3 (APC-CY7), anti-human CD4 (PE-CY7) and anti-human CD8 (BV510) antibodies as well as Annexin V-FITC and PI-PE to detect cell apoptosis. All these cells were analyzed with Beckman CytoFlex S system (Beckman).

Zhang Y., Du L., Wang C., Jiang Z., Duan Q., Li Y., Xie Z., He Z., Sun Y., Huang L., Lu L, & Wen C. (2024). Neddylation is a novel therapeutic target for lupus by regulating double negative T cell homeostasis. Signal Transduction and Targeted Therapy, 9, 18.

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Imaging Innate and Adaptive Immune Responses

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Procedure has been performed as previously described^{69 (link)}. Briefly, WT mice were infected with 10⁴ colony-forming units LmOVA. Mice were given 250 μg brefeldin A by i.p. injection, 6 h before being sacrificed. After 24 h, mice were euthanised, and the spleens were removed, fixed in 4% PFA and cryopreserved in OCT. Serial sections (30 µm in thickness) of frozen spleens were stained overnight at +4 °C in a humidified chamber with the following Abs: anti-B220 PB (1:300; 103230, Biolegend), anti-CD169 Alexa647 (1:300; 142407, Biolegend), anti-CD8 BV510 (1:100; 100751, Biolegend), anti-IFNγ BV421 (1:200; 505829, Biolegend), anti-I-A/I-E biotin (1:200; 107603, Biolegend), or anti-NKp46 (1:200; AF2225, R&D). For secondary detection, sections were then washed and incubated with Streptavidin-Cy3 (1:200; 016-160-084, Jackson Immunoresearch) and anti-goat IgG A647 (1:500; ab150131; Abcam) for 3 h at RT. All sections were analysed by confocal microscopy. Quantification of the interaction between DCs and IFNγ-producing cells was performed manually. DCs were randomly selected in the IFNγ-rich area. For each DC, the number of IFNγ-producing CD8 T cells and the number of IFNγ-producing NK cells was recorded and averaged.

Gajdasik D.W., Gaspal F., Halford E.E., Fiancette R., Dutton E.E., Willis C., Rückert T., Romagnani C., Gerard A., Bevington S.L., MacDonald A.S., Botto M., Vyse T, & Withers D.R. (2020). Th1 responses in vivo require cell-specific provision of OX40L dictated by environmental cues. Nature Communications, 11, 3421.

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Detailed Co-Incubation Assay for Immune Cell Interactions

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For experiments using F5-Jurkat, 1G4-Jurkat, MART1-K562 and NYESO-K562 cells, co-incubations were set up in 96-well U-bottom plates at a ratio of 5:1 Jurkat to K562 (300,000 cells per well) and co-incubated for 45 min at 37 °C. Ratios for co-incubation experiments involving SCT-Jurkat cells and TCR-K562 cells are indicated in the text and figure legends. The co-incubation was then centrifuged for 5 min at 1,500 r.p.m. and the medium was aspirated by vacuum. The cell pellets were resuspended with cold PBS solution containing 2 mM EDTA and then centrifuged. Cells were stained with different antibodies at 4 °C for 20 min, washed twice, and then analyzed by flow cytometry using MACSQuant Analyzer 10 (Miltenyi Biotec). The following antibodies were used to detect expression of surface markers: anti-CD3-PE, anti-CD8-BV510, anti-TCRαβ-PacificBlue, anti-TCRαβ-PE/Cy7, anti-LNGFR-PE, anti-LNGFR-APC, anti-HLA-A2-PacificBlue, anti-HLA-A2-BV510, and anti-muTCRa β-PE/Cy7 (all from Biolegend). Flow cytometry gating strategies are described in the Supplementary Note.

Li G., Bethune M.T., Wong S., Joglekar A.V., Leonard M.T., Wang J.K., Kim J.T., Cheng D., Peng S., Zaretsky J.M., Su Y., Luo Y., Heath J.R., Ribas A., Witte O.N, & Baltimore D. (2019). T cell antigen discovery via trogocytosis. Nature methods, 16(2), 183-190.

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T Cell Subsets Identification and Sorting

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Human T cells were surface stained with anti-CD3 Percp-cy5.5, anti-CD4 FITC, anti-CD8 BV510, anti-CD27 APC, anti-CD45RA PE-Cy 7 (Biolegend). CD4⁺/CD8⁺ T cells were sorted as CD3⁺CD4⁺/CD3⁺CD8⁺ cells. Central memory T cells were stained with CD27⁺CD45RA⁻. Effector memory T cells were stained with CD27⁻CD45RA⁻. Naïve T cells were stained with CD27⁺CD45RA⁺. Terminally T cells were stained with CD27⁻CD45RA⁺.

Li W., Sun X., Wang J., Zhao Q., Dai R., Wang Y., Zhou L., Westerberg L., Ding Y., Zhao X, & Liu C. (2017). Defective thymic output in WAS patients is associated with abnormal actin organization. Scientific Reports, 7, 11978.

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T Cell Immunophenotyping by Flow Cytometry

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We applied BD FACSCalibur and BD FACSAria II instruments for FCM detection. BD FACSDiva software and FlowJo software were used for analysis. FCM detection was performed according to the schedule shown in Figure 2C. CD3, CD4, CD8, and CAR expression were evaluated on days 6, 10, and 14, and memory T subsets were evaluated on days 10 and 14. We used His-tagged recombinant CEACAM5 (CEA-His; Sino Biological Inc., 11077-H08H-50) and biotin-protein L (GenScript, M00097) for specific detection of CAR expression. The following antibodies were used: anti-CD3-PE-Cy7 (BioLegend, 300420), anti-CD4-BUV395 (BD, 564724), anti-CD8-BV510 (BioLegend, 344732), anti-CD25-BV421 (BioLegend, 302630), anti-CD45RA-BV421 (BioLegend, 304130), anti-CD45RO-PerCP-Cy5.5 (BioLegend, 304222), AF-647-conjugated IgG fraction of mouse monoclonal anti-biotin (Jackson, 200-602-211) and anti-CD197-PE (BioLegend, 353204).

Zhang C., Wang L., Zhang Q., Shen J., Huang X., Wang M., Huang Y., Chen J., Xu Y., Zhao W., Qi Y., Li Y., Ou Y., Yang Z, & Qian C. (2023). Screening and characterization of the scFv for chimeric antigen receptor T cells targeting CEA-positive carcinoma. Frontiers in Immunology, 14, 1182409.

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Spleen Harvesting and Immune Cell Analysis

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Spleens were harvested from pups born to ZIKV-immune or naïve mothers at 4- to 5-weeks of age after being humanely euthanized with CO₂. Splenocytes were plated into 96-well round-bottom plates at 1 × 10⁶ cells/well in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% HEPES. Splenocytes were washed with PBS and stained with anti-CD3 PerCP-Cy5.5 (Tonbo Biosciences, cat. #65-0031-U100), anti-CD4 APC eflour780 (Thermo Fisher Scientific, cat. #47-0041-82), and anti-CD8 BV510 (BioLegend, cat. #100751) or with anti-CD19 PE (Thermo Fisher Scientific, cat. #12-0193-83), anti-CD138 PerCP-Cy5.5 (BioLegend, cat. #142510), and anti-mouse IgD FITC (BD Biosciences, cat. #553439). Cells were incubated with these Abs (each at 1:200 dilution) for 30 minutes, followed by washing for 3 times with FACs buffer. The cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, cat. #554714), washed and resuspended in FACs buffer.

Fowler A.M., Tang W.W., Young M.P., Mamidi A., Viramontes K.M., McCauley M.D., Carlin A.F., Schooley R., Swanstrom J., Baric R.S., Govero J., Diamond M.S, & Shresta S. (2018). Maternally Acquired Zika Antibodies Enhance Dengue Disease Severity in Mice. Cell host & microbe, 24(5), 743-750.e5.

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Characterization of 4-1BBL and Calreticulin Expression in Osteosarcoma

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Viral 41BBL expression in osteosarcoma cell lines was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie-NIR Fixable Viability Kit (423105, BioLegend) following the manufacturer's protocol and then with a PE-conjugated anti–4-1BBL antibody.
Calreticulin cell surface expression was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie Green Fixable Viability Kit (423111, BioLegend) and then with a fluorophore-conjugated anti-calreticulin antibody (Abcam).
Fluorescence emission was analyzed using a FACSCanto II system with FACSDiva software (RRID:SCR_001456).
To identify immune cell populations, infiltrating immune cells were surface stained with the following antibody panel: anti–CD45-AF700 (BioLegend), anti–Ly6G-PerCP-Cy5.5 (BioLegend), anti–Ly6C-FITC(BioLegend), anti–F4/80-APC (BioLegend), anti–CD8-BV510 (BioLegend), anti–CD11b-BUV395 (BioLegend), and anti–CD4-BUV496 (BioLegend). PromoFLuor840 maleimide (PromoKine) was used as a viability marker. Samples were acquired with a CytoFlex flow cytometer (Beckman Coulter RRID:SCR_019627) and data analyses were performed using FlowJo v10 (RRID:SCR_008520).

Martinez-Velez N., Laspidea V., Zalacain M., Labiano S., García-Moure M., Puigdelloses M., Marrodan L., Gonzalez-Huarriz M., Herrador G., de la Nava D., Ausejo-Mauleon I., Fueyo J., Gomez-Manzano C., Patiño-García A, & Alonso M.M. (2022). Local Treatment of a Pediatric Osteosarcoma Model with a 4-1BBL Armed Oncolytic Adenovirus Results in an Antitumor Effect and Leads to Immune Memory. Molecular Cancer Therapeutics, 21(3), 471-480.

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Multiparametric Flow Cytometry for Immune Cell Analysis

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Human cells were stained with the following fluorochrome-labeled antibodies: anti-CD45-PECy7 (Biolegend), anti-CD8-BV510 (Biolegend), anti-CD25-BV421 (Biolegend), anti-CD69-PerCPCy5.5 (Biolegend), anti-CD137-Biot (5D1, the laboratory’s own hybridoma), anti-Ki67-AF488 (Biolegend) and anti-Granzyme-AF647 (Biolegend). Streptavidin-PE was added to detect 5D1-Biot Ab. Isotype control mixes was prepared with mIgG1-BV421 (Biolegend), mIgG1-PerCPCy5.5 (Biolegend), mIgG1-AF488 (Biolegend), mIgG1-AF647 (Biolegend). In the case of 5D1-Biot, FMO was performed. M11 CAR expression was verified with anti-mIgG(H+L)-AF647 (Invitrogen) just before stimulation with mesothelin-Fc-beads.
Mouse T cells were stained with: anti-CD8-PECy7 (Biolegend), anti-CD25-APC (Biolegend), anti-CD69-BV510 (Biolegend), anti-CD137-PE (Biolegend), anti-PD1-PerCPCy5.5 (Biolegend), and anti-Ki67-AF488 (BD Bioscience). Rat IgG1-APC (Biolegend), Arm Hamster-BV510 (Biolegend), Syr Hamster-PE (Biolegend), Rat IgG2a-PerCPCy5.5 (Biolegend) and mIgG1-AF488 (Biolegend) antibodies were used as isotype-matched negative controls.
Zombie NiR (Biolegend) was used to exclude cell death. Samples were acquired on a BD Canto II (BD Biosciences) and CytoFlex S systems (Beckman Coulter). Analyses were performed using FlowJo (Tree Star) and CytExpert software (Beckman Coulter).

Glez-Vaz J., Azpilikueta A., Olivera I., Cirella A., Teijeira A., Ochoa M.C., Alvarez M., Eguren-Santamaria I., Luri-Rey C., Rodriguez-Ruiz M.E., Nie X., Chen L., Guedan S., Sanmamed M.F., Luis Perez Gracia J, & Melero I. (2022). Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies. Journal for Immunotherapy of Cancer, 10(3), e003532.

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Flow Cytometry Analysis of Osteosarcoma Cells

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Viral 41BBL expression in osteosarcoma cell lines was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie-NIR™ Fixable Viability Kit (423105, Biolegend, San Diego, CA) following the manufacturer’s protocol and then with a PE-conjugated anti-4-1BBL antibody.
Calreticulin cell surface expression was determined by flow cytometry. Cells were stained first with a cell death detection antibody in the Zombie Green Fixable Viability Kit (423111, Biolegend, San Diego, CA) and then with a fluorophore-conjugated anti-calreticulin antibody (Abcam).
Fluorescence emission was analyzed using a FACSCanto™ II system with FACSDiva software (RRID:SCR_001456).
To identify immune cell populations, infiltrating immune cells were surface stained with the following antibody panel: anti-CD45-AF700 (Biolegend, San Diego, CA), anti-Ly6G-PerCP-Cy5.5 (Biolegend, San Diego, CA), anti-Ly6C-FITC (Biolegend, San Diego, CA), anti-F4/80-APC (Biolegend, San Diego, CA), anti-CD8-BV510 (Biolegend, San Diego, CA), anti-CD11b-BUV395 (Biolegend, San Diego, CA), and anti-CD4-BUV496 (Biolegend, San Diego, CA). PromoFLuor840 maleimide (PromoKine) was used as a viability marker. Samples were acquired with a CytoFlex flow cytometer (Beckman Coulter RRID:SCR_019627) and data analyses were performed using FlowJo v10 (RRID:SCR_008520).

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Immunophenotyping of NK and Senescent T-cells in Early MS

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Peripheral blood mononuclear cells (PBMC) were isolated from blood samples taken in EDTA collection tubes using Lymphoprep, and subsequently cryopreserved in fetal calf serum with 10% DMSO. The gating strategy of the study is shown in Figure 1. NK-cell immunophenotype was evaluated in early MS patients (n=120) as previously reported [12] , using the following conjugated monoclonal antibodies: anti-CD3-PerCP, anti-CD56-APC (BD Biosciences), NKG2C-PE (R&D Systems), and DAPI. A selected panel of immunological markers of differentiated /senescent T-cells previously related to CMV infection (CD27-CD28-/CD57+/LILRB1+ T-cells) [16] [17] [18] were evaluated in a subcohort of MS patients (early MS, n=35; non-early MS, n=32) and controls (n=32) stratified for CMV serology. Samples were stained by indirect immunofluorescence with LILRB1 HP-F1and anti-mouse Ig-PE-Cy7 (Biolegend), washed and further incubated with anti-CD3-APC-H7, anti-CD27-PERCP-Cy5.5, anti-CD28-PE-CF594, CD57-PE (BD Biosciences), and anti-CD4-FITC (eBioscience), anti-CD8-BV510 (Biolegend). All samples were analyzed at the Flow Cytometry Unit (UPF/CRG, Barcelona) with an LSRII-Fortessa flow cytometer (BD Biosciences).

Alari-Pahissa E., Moreira A., Zabalza A., Alvarez-Lafuente R., Munteis E., Vera A., Arroyo R., Alvarez-Cermeño J.C., Villar L.M., López-Botet M, & Martínez-Rodríguez J.E. (2018). Low cytomegalovirus seroprevalence in early multiple sclerosis: a case for the 'hygiene hypothesis'?. European journal of neurology, 25(7).

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