Cells were removed from plates by gentle trypsinization with 0.02% trypsin (Worthington Biochemical) in HBS Ca (20 mM Hepes, 137 mM NaCl, 3 mM KCl, 1 mM CaCl, pH 7.4) for 3–4 minutes at 37°C to minimize cadherin-11 proteolysis. Cadherin-11 surface expression was analyzed by flow cytometry (CytoFlex, Beckman Coulter) using ZombieAqua Live/Dead stain (BioLegend) and anti-cadherin-11 23C6 primary antibody as previously described[13 (link)]. Data was analyzed using Kaluza software.
Zombie aqua live dead stain
Manufactured by BioLegend
Zombie Aqua live/dead stain is a fluorescent dye used to distinguish between live and dead cells in a sample. It emits fluorescence upon binding to cellular components, allowing for the detection and quantification of live and dead cells.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex, by Beckman Coulter (1 mentions)
Brefeldin a bfa, by Merck Group (1 mentions)
Cd11b pe cy7, by BioLegend (1 mentions)
Fortessa, by BD (1 mentions)
Cytofix, by BD (1 mentions)
Accutase, by BioLegend (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Cd3 sk7, by BD (1 mentions)
Cd4 sk3, by BD (1 mentions)
Cd45ra hi100, by BD (1 mentions)
Cd56 hcd56, by BioLegend (1 mentions)
8 rpa t8, by BD (1 mentions)
Cd27 o323, by Thermo Fisher Scientific (1 mentions)
Foxp3 pch101, by Thermo Fisher Scientific (1 mentions)
Fortessa x20 flow cytometer, by BD (1 mentions)
Fc block human trustain fcx, by BioLegend (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Facs buffer, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Fc block, by BD (1 mentions)
6 protocols using zombie aqua live dead stain
1
Cadherin-11 Surface Expression Analysis
2
Intracellular Cytokine Staining of RAW264.7 Cells
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RAW264.7 cells of BMDMs were collected after washing in ice-cold PBS and then detaching the cells with Accutase (BioLegend). Prior to collection, cells were incubated in 10ug/ml of Brefeldin A (BFA, Sigma). BFA was added to the culture four hours prior to harvest for staining.
Cells and all reagents were maintained at 4°C throughout the intra-cellular staining protocol. Harvested cells were washed twice in PBS and re-suspended in approximately 50ul of PBS. Cells were stained with 100ul of 1:1000 Zombie Aqua live/dead stain (BioLegend) in PBS on ice for 8–10 minutes in the dark. F
c receptors were blocked with 5ul of 2mg/ml rat IgG for five minutes. Cells were fixed with BD Cytofix and permeabilized with BD Cytoperm. Intracellular staining was performed with the cocktail of antibodies made in permeability buffer. BV421-TNF (MP6-XT22; BioLegend), APC-IL6 (MP5-20F3; BioLegend, eFluor 610-NOS2 (CXNFT; ThermoFisher Scientific), PE-pro-IL-1β (NJTEN3, ThermoFisher Scientific), FITC-F4/80 (BM8, BioLegend) and PE-Cy7 CD11b (M1/70, BioLegend) were used for staining RAW264.7 cells. BMDMs and RAW264.7 cells were pre-gated on live cells, singlets, forward scatter, and side scatter (for gating intact cells), F4/80+ and/or CD11b. Flow cytometry was performed on a BD Fortessa and the analysis done using FlowJo (v10.7.2).
Cells and all reagents were maintained at 4°C throughout the intra-cellular staining protocol. Harvested cells were washed twice in PBS and re-suspended in approximately 50ul of PBS. Cells were stained with 100ul of 1:1000 Zombie Aqua live/dead stain (BioLegend) in PBS on ice for 8–10 minutes in the dark. F
c receptors were blocked with 5ul of 2mg/ml rat IgG for five minutes. Cells were fixed with BD Cytofix and permeabilized with BD Cytoperm. Intracellular staining was performed with the cocktail of antibodies made in permeability buffer. BV421-TNF (MP6-XT22; BioLegend), APC-IL6 (MP5-20F3; BioLegend, eFluor 610-NOS2 (CXNFT; ThermoFisher Scientific), PE-pro-IL-1β (NJTEN3, ThermoFisher Scientific), FITC-F4/80 (BM8, BioLegend) and PE-Cy7 CD11b (M1/70, BioLegend) were used for staining RAW264.7 cells. BMDMs and RAW264.7 cells were pre-gated on live cells, singlets, forward scatter, and side scatter (for gating intact cells), F4/80+ and/or CD11b. Flow cytometry was performed on a BD Fortessa and the analysis done using FlowJo (v10.7.2).
3
CARMIL2-expressing T cell analysis
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We identified the T cell populations in which CARMIL2 reexpression occurred by staining PBMCs with the Abs against the following: CD3 (SK7), CD4 (SK3), CD8 (RPA-T8), CD25 (M-A251, all from BD), CD27 (O323; eBioscience), CD45RA (HI100, BD), CD56 (HCD56; Biolegend), FoxP3 (PCH101; eBioscience), and CARMIL2 (EM53; Exbio). Viability was assessed with Zombie Aqua Live/Dead stain (Biolegend). In patients with CARMIL2-expressing T cell populations, T lymphoblasts were generated by stimulating 106 PBMCs with 5 ng/ml PMA, 1 µM ionomycin, and 100 U/ml IL-2 for 2 d before further expansion for 8–16 d in complete RPMI 1640 supplemented with 100 U/ml IL-2. The T lymphoblasts were sorted with Abs against CD3 (SK7), CD4 (SK3), CD8 (RPA-T8, all from BD), and CARMIL2 (EM53; Exbio). DNA was extracted from the CARMIL2-expressing cell populations with the DNeasy Blood and Tissue kit (Qiagen), and reversion events were confirmed by Sanger sequencing.
4
Multiparametric Flow Cytometry of Whole Blood
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Staining was performed on whole blood. 100 µl of blood was washed in PBS and incubated with Zombie Aqua live/dead stain (product number 423101; BioLegend) at room temperature for 10 min. Samples were incubated with FC-Block (Human TruStain FcX, product number 422302; BioLegend) in staining buffer (PBS, 5 mM EDTA, 0.5% BSA), followed by addition of primary antibodies in staining buffer and incubation on ice for 20 min. Cells were washed and incubated with fluorophore-conjugated streptavidin on ice for 15 min. Erythrocytes were lysed with chilled ACK lysis buffer (product number A10492-01; Gibco), and cells were fixed in 4% PFA for 20 min. Cells were resuspended in staining buffer and analysed on a BD X20 Fortessa flow cytometer. Data were analysed using FlowJo (FlowJo, LLC). See Table S2 for flow cytometry reagents.
Table S2
5
Optimizing Live/Dead Cell Staining
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Live/dead was titrated to achieve the best staining index, which was 1:500 for 20 minutes in PBS, and then quenched with two washes of FACS buffer (BD Biosciences, Franklin Lake, NJ); Other concentrations tested were 1:100, 1:300, and 1:1000. The lot of Zombie Aqua live/dead stain (Biolegend, San Diego, CA) was coordinated across study sites.
6
Cytokine Profiling via Intracellular Staining
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For intracellular cytokine staining (ICS), cells were cultured with 50 ng PMA (phorbol 12-myristat-13-acetate), 500 ng ionomycin and 3 mM monensin (all from Sigma-Aldrich) for 4 hours prior to flow cytometry analysis (7, 8, 15) . Prior to immune-labelling, cells were treated with Fc block (BD Biosciences) to reduce nonspecific Ab binding. Cells were stained for cell surface markers, then fixed and permeabilised in Cytofix/Cytoperm (BD Biosciences) before intracellular detection of cytokines. Flow cytometric analysis of cells was performed using Zombie Aqua live/dead stain (BioLegend), anti-CD4 (RM4-5), anti-IFN-g (XMG 1.2), and anti-IL-17 (TC11-18H.1). Cell reads were acquired using a FACSCanto II flow cytometer using FACSDiva software (BD Biosciences). Data analysis was conducted using FlowJo Version 10.7.1 (Tree Star Inc, US).
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