Cultured cells were washed with PBS, stained with Zombie Fixable Viability Kit (Biolegend), followed by staining with monoclonal Abs against special surface markers. For cell memory state analysis, cells were incubated with PB-anti-CD3 (SP34-2, BD), FITC-anti-Vγ2 (7A5, Thermo Scientific), PE-anti-Vδ2 (B6, Biolegend), BV785-anti-CD45RA (HI100, Biolegend), PE/Cy7-anti-CD27 (O323, Biolegend), PE/Cy5-anti-CD28 (CD28.2, Biolegend) for 20 min at room temperature in dark. For phenotyping of special surface markers, cells were incubated with PB-anti-CD3 (SP34-2, BD), FITC-anti-Vγ2 (7A5, Thermo Scientific), PE-anti-Vδ2 (B6, Biolegend), APC-anti-CCR5 (J418F1, Biolegend), PE/Cy5.5-anti-LFA-1 (TS1/18, Biolegend) for 20 min at room temperature in dark. Then cells were washed and fixed by fixing buffer (2% formalin in PBS), analyzed on an LSR Fortessa flow cytometer (BD).
Pe anti vδ2 b6
Manufactured by BioLegend
Sourced in United States
PE-anti-Vδ2 (B6) is a monoclonal antibody conjugated with Phycoerythrin (PE) that binds to the Vδ2 chain of the T cell receptor on human T cells. It is designed for use in flow cytometry applications to identify and quantify Vδ2-expressing T cells within a sample.
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Lab products found in correlation
Zombie fixable viability kit, by BioLegend (3 mentions)
Fitc anti vγ2 7a5, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Sp34 2, by BD (1 mentions)
Pe cy7 anti tnf α mab11, by BioLegend (1 mentions)
Macs separation column, by Miltenyi Biotec (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
4 protocols using pe anti vδ2 b6
1
Multiparametric Immune Cell Profiling
2
Measuring Mycobacterial Immunity in PBMCs
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PBMCs cultured for 7 days were firstly treated with/without 40 ng/ml HMBPP, pulsing PE/CF594-anti-CD107a (H4A3, Biolegend), and BFA (GolgiPlug, BD) for 6 h. Then cells were stained with Zombie Fixable Viability Kit (Biolegend), incubated with PB-anti-CD3 (SP34-2, BD), FITC-anti-Vγ2 (7A5, Thermo Scientific), PE-anti-Vδ2 (B6, Biolegend) for 20 min at room temperature in dark. Cells were permeabilized for 30 min at 4 degrees (Cytofix/Cytoperm, BD). After wash, cells were incubated with BV711-anti-IFN-γ (4S.B3; Biolegend), PE/Cy7-anti-TNF-α (Mab11, Biolegend), Percp/Cy5.5-anti-GM-CSF (BVD2-21C11, Biolegend) for 30 min at room temperature in dark. Then cells were washed and analyzed on an LSR Fortessa flow cytometer (BD). CD107a was used to assess degranulation of cytotoxic molecules; IFN-γ, TNF-α, and GM-CSF was used to evaluating anti-mycobacteria effector function. Flow data were analyzed by FlowJo (TreeStar).
3
Vγ2Vδ2 T Cells Inhibit BCG Survival
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M.bovis BCG-infected A549 cells were prepared as target cells at MOI = 10 as we previously described [17 (link),18 (link)]. To isolate Vγ2Vδ2 T cells, macaque PBMCs in ZOL + IL-2 cultures were stained with PE-anti-Vδ2 (B6, Biolegend), and then incubated with anti-PE microbeads (Miltenyi Biotech). Vδ2+ T cells were then isolated using MACS Separation columns (Miltenyi Biotech) according to manufacturer’s protocol, serving as effector cells. The B cells were isolated from PBMCs with CD19+ B cells isolation kit (Miltenyi Biotech) according manufacturer’s protocol. BCG-infected A549 cells were cultured with media alone or with purified Vδ2+ or CD19+ cells at a ratio of E: T = 10: 1 in 96-well plates for 3 days. Mycobacteria viability was quantified via counting CFU as previously described [19 (link)].
4
Vγ2Vδ2 T Cell Cytokine Profiling
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Flow cytometry was performed as described in the previous reports (31 (link), 32 (link)). H1975 cells were firstly plated in a 24-wells plate. On the next day, expanded and negatively enriched Vγ2Vδ2 T cells were added into each of the wells with doses of Y111 or CD3 Isotype together with BV510-anti-CD107a (H4A3, Biolegend, San Diego, USA) and BFA (Golgi Plug, BD, San Jose, USA). After co-cultured for 6 hours, the cells were stained with Zombie Fixable Viability Kit (Biolegend, San Diego, USA), followed by incubation with APC-anti-CD3 (SP34-2, BD, San Jose, USA), PE-anti-Vδ2 (B6, Biolegend, San Diego, USA) for 20 min at room temperature in dark. The cells were permeabilized for 30 min at 4 degrees (Cytofix/Cytoperm, BD, San Jose, USA). After wash, the cells were incubated fixation buffer with BV650-anti-IFNγ (4S.B3; Biolegend, San Diego, USA), BV421-anti-TNFα (Mab11, Biolegend, San Diego, USA) for 30 min at room temperature in dark. Then cells were washed and collected by a BD FACSelesta flow cytometry.
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