Freshly isolated PBMCs or TILs (1 × 106) were washed and incubated with fluorophore-conjugated monoclonal antibodies including Alexa Fluor 750-anti-CD45, Alexa Fluor 700-anti-CD3, BV421-anti-TCRγδ, FITC-anti-Vδ1, and PE-anti-Vδ2 (all from Biolegend, San Jose, CA, USA) for 20 min at room temperature in the dark. For intracellular cytokine detection, PBMCs or TILs (1 × 106) were resuspended in RPMI 1640 medium supplemented with 10% FBS (Gibco) and stimulated with phorbol-12-myristate 13-acetate (50 ng/ml), ionomycin (1 μg/ml), and brefeldin (1 μg/ml) (all from Biogems, Rocky Hill, NJ, USA) for 5 h in 5% CO2 atmosphere at 37 °C. Cells were then washed, fixed, permeabilized, and stained with APC-anti-IL-17A, and APC/cy7-anti-IFN-γ (Biolegend, San Jose, CA, USA) according to the manufacturer’s protocol. Fluorescence data were collected on a FACS Aria II (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, US).
Pe anti vδ2
Manufactured by BioLegend
Sourced in United States
PE-anti-Vδ2 is a fluorochrome-conjugated antibody that binds to the Vδ2 chain of the T cell receptor on γδ T cells. It is suitable for flow cytometric analysis of Vδ2-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 750 anti cd45, by BioLegend (2 mentions)
Fitc anti vδ1, by BioLegend (2 mentions)
Alexa fluor 700 anti cd3, by BioLegend (2 mentions)
Bv421 anti tcrγδ, by BioLegend (2 mentions)
Facsaria 2, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Anti il 17a apc, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Biogems (1 mentions)
Phorbol 12 myristate 13 acetate, by Biogems (1 mentions)
Facsdiva software, by BD (1 mentions)
Anti pd 1 bv711, by BioLegend (1 mentions)
Anti cd3 pe cy7, by BioLegend (1 mentions)
Lsrfortessa cell analyser, by BD (1 mentions)
Bv421 anti cd127, by BioLegend (1 mentions)
Golgiplug, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti cd4 pe cy7, by BioLegend (1 mentions)
Il 17a pe cy7, by BioLegend (1 mentions)
Anti cd8 apc cy7, by BioLegend (1 mentions)
5 protocols using pe anti vδ2
1
Immunophenotyping of γδ T Cells
2
Multiparameter Immune Profiling of Bone Marrow
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Briefly, fresh bone marrow samples were stained with the following fluorochrome-labeled antibodies: PE-Cy7 anti-CD3, BV510 anti-TCRαβ, PE anti-Vδ2, APC-Cy7 anti-NKG2D, BV711 anti-PD-1, PE-Cy5 anti-CD25, and BV421 anti-CD127 (BioLegend, USA), and FITC anti-Vδ1 (Thermo Scientific, USA). Polychromatic flow cytometric analyses were performed on a BD LSRFortessa™ Cell Analyser and further analyzed using BD FACSDiva™ software.
3
Profiling γδ T Cell Responses to Ovarian Cancer
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γδ T cells (1 × 105) sorted from healthy PB were cultured with OC tissues supernatants, BOT tissues supernatants, and RPMI 1640 control medium in 24-well plates in 37 °C at 5% CO2. After 5 days, γδ T cells were harvested and stained with Alexa Fluor 750-anti-CD45, Alexa Fluor 700-anti-CD3, BV421-anti-TCRγδ, FITC-anti-Vδ1, and PE-anti-Vδ2 (all from Biolegend, San Jose, CA, USA) to analysis the levels of Vδ1 T cells and Vδ2 T cells by flow cytometry.
4
Multiparameter Flow Cytometry Analysis
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The following antibodies (Abs) were used for short-term culture or surface marker and intracellular cytokine staining for flow cytometry (all Abs were from Biolegend): anti-CD3-PerCP-cy5.5 (Clone UCHT1), anti-CD8-APC-Cy7 (Clone SK1), anti-CD4-PE-Cy7 (Clone RPA-T4), anti-Vδ1-APC (Clone REA173), anti-Vδ2-PE (Clone B6), anti-Vγ2-FITC (Clone 7A5), anti-TNF-α-PE (Clone MAb11), anti-IFN-γ-PE-Cy7 (Clone 4S.B3), anti-interleukin-17A (IL-17A)-PE-Cy7 (Clone BL168), anti-Granzyme A-PE (Clone CB9), anti-Perforin-APC (Clone dG9), anti-granulocyte macrophage colony-stimulating factor (GM-CSF)-APC (Clone BVD2-21C11). The isotype control mAbs were purchased from the related company, respectively. The reagents listed below were all commercial products: brefeldin A (GolgiPlug, BD Biosciences), Cytofix/Cytoperm, and Perm buffer (BD Biosciences). The isotype control mAbs were purchased from the related company, respectively.
5
Multiparametric Flow Cytometric Profiling
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Peripheral blood mononuclear cells were incubated with a mix of monoclonal antibodies consisting of anti-CD3 APC (BioLegend, San Diego, USA; cat. No. 300439, clone: UCHT1), anti-TCRγδ BV421 (BD Biosciences, Warsaw, Poland; cat. No. 744870, clone: 11F2), anti-Vδ1 FITC (ThermoFisher, Warsaw, Poland; cat. No. TCR2730, clone: TS8.2), anti-Vδ2 PE (BioLegend; cat. No. 331408, clone: B6), anti-CTLA-4 APC-Fire750 (BioLegend; cat. No. 349930, clone: L3D10), anti-PD-1 PE-Cy7 (BD Biosciences, Franklin Lakes, USA; cat. No. 561272, clone: EH12.1), anti-TIGIT BV650 (BD Biosciences; cat. No. 747840, clone: 741182), anti-NKp30 BV785 (BioLegend; cat No. 325230, clone: P30-15), and anti-CD226 BV605 (BD Biosciences; cat. No. 742495, clone: DX11). Fluorescence minus one (FMO) controls were used to set the correct gates for each fluorophore. Samples were acquired on a Cytoflex LX (Beckman Coulter, Warsaw, Poland) and analyzed with FlowJo 10 (BD Biosciences). The percentage of positive cells was a standard measure for each of the markers except for CD226, where mean fluorescence intensity (MFI) was additionally provided due to its high overall expression.
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