Single-cell suspensions were treated with FcR-blocking media containing 2.4G2 antibodies. The cells were then stained with fluorescent antibodies on ice for 25 min followed by incubation with cell viability dye (eBioscience). For detection of insulin-binding B cells, cells were incubated with 50 ng/ml biotinylated insulin (Eagle Biosciences) with or without inhibition with 500 ng/ml unconjugated insulin for 30 min on ice followed by staining with APC-conjugated streptavidin (BioLegend) for 15 min. The FoxP3 staining was performed using a transcription factor buffer set (eBioscience). The flow cytometry samples were collected using a FACSCanto II (BD), and data were analyzed using FlowJo software (Tree Star). Cell sorting was performed using a FACSAria II (BD).
Cell viability dye
Manufactured by Thermo Fisher Scientific
The cell viability dye is a fluorescent reagent used to assess cell health and measure cell viability in cell-based assays. It functions by selectively staining dead or dying cells, allowing for the quantification of viable cells in a population.
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Lab products found in correlation
Anti cd45, by Thermo Fisher Scientific (2 mentions)
Transcription factor buffer set, by Thermo Fisher Scientific (1 mentions)
Apc conjugated streptavidin, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Facscanto 2 device, by BD (1 mentions)
Anti cd8, by Thermo Fisher Scientific (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
2 protocols using cell viability dye
1
Single-cell Analysis of Insulin-Binding B Cells
2
Multiparametric Flow Cytometry of Immune Cells
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Surface staining with anti-CD4, anti-CD3, anti-CD8, anti-CD25, anti-CD45.1, and anti-CD45.2 (all eBioscience) was performed as described previously [27] . Intracellular Foxp3, pS6, and p-mTOR staining was performed with a FOXP3 staining buffer set (eBioscience, Germany). Mitochondrial mass measurements were performed with 500 nM MitoTracker (ThermoFisher Scientific). Glucose uptake was determined by means of a glucose uptake cell-based kit (Cayman Chemical) according to manufactures instructions. Cell viability dye (eBioscience) and fluorochrome conjugated Annexin-V (eBioscience) were used to identify apoptotic cells. Flow cytometry was carried out using the FACSCanto II device (BD Biosciences, Germany). Data analysis was performed using FCS Express software.
For gating strategies applied to the data, please refer to Supporting Information Fig. 3 as indicated in the figure captions. We have adhered to the Guidelines for the use of flow cytometry and cell sorting in immunological studies [28] .
For gating strategies applied to the data, please refer to Supporting Information Fig. 3 as indicated in the figure captions. We have adhered to the Guidelines for the use of flow cytometry and cell sorting in immunological studies [28] .
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