Anti cd8 buv805

Manufactured by BD

Anti-CD8 BUV805 is a fluorescent-conjugated antibody used for the detection and analysis of CD8-positive cells in flow cytometry applications.

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5 protocols using anti cd8 buv805

TIL-Tumor Cell Co-culture Assay

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Fresh or defrosted REP TILs were counted and pre-stained in fluorescence-activated cell sorter (FACS) buffer with anti-CD4 BUV496 and anti-CD8 BUV805 (BD, Vianen, the Netherlands) for 30 min at 4°C. Cells were washed once with FACS buffer, and once with 20/80 T-cell mixed media. A total of 1 × 10⁵ live expanded TILs were co-cultured with 2 × 10⁵ live tumor digest cells for 6 h at 37°C. Alternatively, expanded TILs were stimulated with 10 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin (Sigma-Aldrich), or cultured with T cell mixed media alone. After 1 h of co-culture, 1x Brefeldin A and 1x Monensin (Invitrogen, Landsmeer, the Netherlands) were added.

Castenmiller S.M., de Groot R., Guislain A., Monkhorst K., Hartemink K.J., Veenhof A.A., Smit E.F., Haanen J.B, & Wolkers M.C. (2022). Effective generation of tumor-infiltrating lymphocyte products from metastatic non-small-cell lung cancer (NSCLC) lesions irrespective of location and previous treatments. Immuno-Oncology and Technology, 15, 100090.

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Siglec-15 Binding Assay with T Cells

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For blocking assays, T cells were preincubated with anti-human CD11b antibodies (clone M1/70, 557394, BD Biosciences or clone CBRM1/5, 14-0113-81, Thermo Fisher) or matched isotype controls (rIgG2a, 14-4321-82, Thermo Fisher and mIgG1, 14-4714-82, Thermo Fisher) at 10 µg/mL in 2 % BSA in PBS for 30 min at 4 °C. After washing, cells were incubated with recombinant Siglec-15-Fc, Siglec-15-Fc R143A mutant or human IgG1 Fc control (110-HG-100, R&D) at 4 µg/mL for 30 min at 4 °C. Cells were then washed and incubated with anti-human IgG Fc PE (12-4998-82, Thermo Fisher, 1:200), anti-CD4 BUV395 (563550, BD Biosciences, 1:200) and anti-CD8 BUV805 (612889, BD Biosciences 1:200) and incubated for 30 min at 4 °C in the dark. Cells were washed and resuspended in 200 µL 2 % BSA in PBS with DAPI (1:10,000) before acquisition on a FACSymphony.

Lenza M.P., Egia-Mendikute L., Antoñana-Vildosola A., Soares C.O., Coelho H., Corzana F., Bosch A., Manisha P., Quintana J.I., Oyenarte I., Unione L., Moure M.J., Azkargorta M., Atxabal U., Sobczak K., Elortza F., Sutherland J.D., Barrio R., Marcelo F., Jiménez-Barbero J., Palazon A, & Ereño-Orbea J. (2023). Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b. Nature Communications, 14, 3496.

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Multicolor Flow Cytometry Compensation Beads Protocol

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UltraComp eBeads^TM Compensation Beads (Thermofisher) were used to optimize fluorescence compensation settings for multi-color flow cytometric analysis at a Symphony flow cytometer. UltraComp eBeads^TM were stained with the following fluorochrome-labeled anti-human antibodies: anti-CD8–BUV805 (1:200, clone SK1), anti-CD4–BUV496 (1:50, clone SK3), anti-CD86–BUV737 (1:50, clone 2331 FUN-1), anti-CD141–BUV615-P (1:50, clone 1A4), anti-CD56–BUV563 (1:50, clone NCAM 16.2), anti-CD16–BUV395 (1:50, clone 3G8), anti-CD123–BB660-P (1:50, clone 7G3), anti-CD80–BB630 (1:50,clone L307.4), anti-CD21–BV785 (1:50, clone B-ly4), anti-CD27–BV750-P (1:40,clone L128), anti-BAFF-R–BV650 (1:50, clone 11C1), anti-CD94–BV605 (1:50, clone HP-3D9), anti-CD40–APC-R700 (1:50, clone 5C3) (all BD bioscience); anti-CD3–PerCP-Vio700 (1:50, clone REA613) (Miltenyi Biotec); anti-CD57–FITC (1:100, clone TB01), anti-CD14–PE-Cy5.5 (1:200, clone TuK4), fixable viability dye eFluor780 (1:1000) (all eBioscience); anti-CD24–BV711 (1:50, clone ML5), anti-CD19–BV510 (1:25, clone HIB19), anti-HLA-DR–BV570 (1:40, clone L243), anti-IgM–BV421 (1:100, clone MHM-88), anti-CD11c–APC (1:40, clone 3.9), anti-CD38–PE/Dazzle 594 (1:100, clone HB-7), anti-CD10–PE-Cy5 (1:50, clone HI10a), and anti-IgD–PE-Cy7 (1:100, clone IA6-2) (all BioLegend).

Roca C.P., Burton O.T., Gergelits V., Prezzemolo T., Whyte C.E., Halpert R., Kreft Ł., Collier J., Botzki A., Spidlen J., Humblet-Baron S, & Liston A. (2021). AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data. Nature Communications, 12, 2890.

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Comprehensive Immunophenotyping of PBMC Subsets

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Frozen PBMCs from human healthy donors were processed as for the HS1 datasset and stained with live/dead marker and fluorochrome-conjugated antibodies against the following surface markers: anti-CD8-BUV805, anti-CD4-BUV496, anti-CD95-BUV737, anti-CD28-BB660-P, anti-ICOS-BB630, anti-CXCR3-BV785, anti-PD-1-BV750-P, anti-CXCR5-BV650, anti-CCR2-BV605, (all BD Biosciences); anti-CD3-PerCP-Vio700 (Mil-tenyi Biotec); anti-CD45RA-FITC, anti-CD14-PE-Cy5.5, fixable viability dye eFluor780 (all eBioscience); anti-CD25-BV711, anti-CD31-BV480, anti-HLA-DR-BV570, anti-CD127-BV421, anti-CCR4-PE/Dazzle 594, anti-CCR7-PE-Cy7 (all BioLegend).
Samples were stained for 60 min at 4

Roca C.P., Burton O.T., Prezzemolo T., Whyte C.E., Halpert R., Kreft Ł., Collier J.H., Botzki A., Špidlen J., Humblet‐Baron S., & Liston A. (2020). AutoSpill: A method for calculating spillover coefficients to compensate or unmix high-parameter flow cytometry data.

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Comprehensive Immune Cell Phenotyping

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Cells were stained according to the manufacturer's instructions. Briefly, cells were stained with anti-CD69 BUV395 (BD, clone FN50) on the surface and washed prior to stimulation. After the stimulation, cells were surface stained with anti-CD103 BV711 (Biolegend, Ber-ACT8), anti-CD45RA BUV563 (BD, clone HI100), anti-CD27 BV650 (Biolegend, clone 0323), anti-PD-1 PE-Cy7 (BD, clone EH12.2H7), and anti-CD39 BV510 (Biolegend, clone A1). Cells were then fixed and permeabilized with the Foxp3 Staining Buffer Set (Thermofisher), followed by intracellular staining of the cells with anti-CD3 BV605 (eBioscience, clone OKT3) or BUV661 (BD, clone UCHT1), anti-CD4 BUV737 (BD, clone SK3), anti-CD8 BUV805 (BD, clone SK1), anti-CD40L PeDazzle594 (Biolegend, clone 24-31), anti-CD137 AF647 or PeCy7 (Thermofisher Scientific, clone 4B4-1), anti-TNF PeCy7 or FITC (BD, Mab11), anti-IFNγ eF450 or BV785 or Pe (Biolegend, clone 4S.B3), anti-IL17a BV605 (Biolegend, clone BL168), and anti-IL-2 BB700 or APC (BD, clone MQ1-17H12). Samples were analyzed in 0.5% FCS PBS and acquired on a BD FACSymphony. Data analysis was performed using FlowJo (TreeStar, Version 10.0.7).

Oja A.E., Morgana F., Hagen R., Ghandour C.A., Nolte M.A., Rooijakkers S.H.M., Lier R.A.W.v., Bardoel B.W., & Hombrink P. (2022). Polyfunctional pathogen-specific CD4⁺ T cells reside in the lungs and tumors of NSCLC patients.

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