Thawed PBMC were cultured in the presence of infectious VZV (120,000 pfu/ml) or medium control for 2 days, or stained with Cell Trace Violet (Biolegend) and cultured in the presence of VZV cell lysate or control for 6 days. Blocking experiments were set up with 10ug/ml anti-PDL1 (Biolegend, clone 29E.2A3), 10 µg/ml anti-PD1 (Biolegend clone EH12.2H7), 10 µg/ml anti-TIM-3 (Biolegend clone F38-2E2) and/or LAG-3 Fc chimera (R&D Systems), 2µg/ml. Brefeldin A was added for the last 16 h of virus stimulation. PBMC were washed with PBS and incubated with Zombie Yellow viability stain (Biolegend). Cells were washed, stained and analyzed as above.
Zombie yellow viability stain
Manufactured by BioLegend
Zombie Yellow is a viability stain that can be used to detect dead cells. It is a fluorescent dye that binds to proteins in dead cells, emitting a yellow fluorescent signal that can be detected using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti pd 1, by BioLegend (1 mentions)
Anti tim 3, by BioLegend (1 mentions)
Celltrace violet, by BioLegend (1 mentions)
Anti pd l1, by BioLegend (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Anti mouse cd38, by BioLegend (1 mentions)
Trustain fcx antibody, by BioLegend (1 mentions)
Negative selection b cell isolation kit, by STEMCELL (1 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
Hcd14, by BioLegend (1 mentions)
Aria 3, by BD (1 mentions)
4 protocols using zombie yellow viability stain
1
Investigating VZV-Specific T Cell Responses
2
Phagocytosis of Apoptotic K562 Cells
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To stimulate apoptosis, K562 cells were exposed to UV light for 15 min. Apoptotic K562 cells were stained with ZombieYellow viability stain (Biolegend), after which apoptosis was confirmed with >90% of cells staining positive. Culture media was removed from Mϕ cultures and target cells in RPMI + 10% FCS were added at a ratio of 2:1 (K562). Culture plates were centrifuged at 450 g for 2 min to synchronize phagocytosis. Following 1 h of incubation at 37°C, cells were washed and labeled with BV711 mouse anti-human CD14 antibody (BD Biosciences), and analyzed using the BD Biosciences LSR Fortessa cell analyzer.
3
Isolation and Sorting of Germinal Center B Cells
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GC B cells were generated and sorted using a previously described protocol (Hwang et al., 2015 (link)). Briefly, 6–12-week-old M2A-GFP KI mice were immunized with sheep’s red blood cells. After 8–10 days, total B cells from the spleens and lymph nodes were isolated using the Negative Selection B cell isolation kit (StemCell Technologies) according to the manufacturer’s instructions. Dead cells were stained using Zombie Yellow viability stain (BioLegend) and Fc receptors were blocked with the mouse TruStain FcX antibody (#156604). Cells were immunostained with anti-mouse CD38 (#102719), B220 (#103235), and GL-7 (#144617) purchased from BioLegend. GC B cells were sorted on a BD Aria III FACs sorter (Beckton Dickinson) for GFP+, Zombie Yellow-, B220+, CD38low and GL-7+ cells, and were used immediately.
4
Isolation and Functional Evaluation of Regulatory T Cells
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Tregs were sorted from SVF and peripheral blood (PB) based on positive viability and the following markers: Lin-CD4 + CD25 + CD127dim using an Aria III (BD) FACS sorter. Cells were stained with antibodies against CD14 (HCD14; Catalog #325604), CD15 (W6D3; #323004), CD19 (HIB19; #302206), CD20 (2H7; #302304), CD56 (HCD56; #318304); TIGIT (A15153G; #372716); CD25 (M-A251; #356110); CD8 (SK1; #344724); CD127 (A019D5; #351332); CTLA-4 (BNI3; #369609); OX-40 (Ber-ACT35; #350012); PD-1 (EH12.2H7; #329949); CD4 (RPA-T4; #300530); CD3 (OKT3; #317330); Zombie Yellow Viability Stain all from BioLegend, with validation per the manufacturer’s website (https://www.biolegend.com/en-us/quality/quality-assurance-certificates ). Additionally, cells were stained with Foxp3 (PCH101; #14-4776-82) from Invitrogen. Gating was determined based on staining antibody comparison to isotype control antibody.
To assess Treg function, previously separated PBMCs from the same patient were labeled with CellTrace CFSE (Invitrogen) and then cultured in the absence or presence of isolated blood or VAT Tregs (2 PBMC:1 Treg). Cells were cultured in a 96-well round bottom plate at 10,000 cells/well in the presence of one Treg Inspector Bead (Miltenyi) per 2 cells. After 4 days of culture, cells were stained for CD3 and viability and flow performed on an Aurora (Cytek).
To assess Treg function, previously separated PBMCs from the same patient were labeled with CellTrace CFSE (Invitrogen) and then cultured in the absence or presence of isolated blood or VAT Tregs (2 PBMC:1 Treg). Cells were cultured in a 96-well round bottom plate at 10,000 cells/well in the presence of one Treg Inspector Bead (Miltenyi) per 2 cells. After 4 days of culture, cells were stained for CD3 and viability and flow performed on an Aurora (Cytek).
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