Cell-surface expression of GD2 (cat#565991, BD Biosciences), CD8 (cat#301041, BioLegend) and CD171 (cat#130–100-691, Miltenyi Biotec) was detected by fluorophore-conjugated monoclonal antibodies. EGFRt expression was detected using biotinylated cetuximab (Bristol-Myers Squibb) and a phycoerythrin (PE)-conjugated streptavidin antibody (cat#12–4317-87, BioLegend). Activation and exhaustion were assessed by fluorophore-conjugated monoclonal antibodies detecting CD137 (cat#309819, BioLegend), CD25 (cat#302622, BioLegend), PD1 (also known as PDCD1 or CD279, cat#329922, BioLegend), TIM3 (cat#345006, Biolegend) and LAG3 (cat#565721, BD Biosciences). Flow cytometry was performed on a Fortessa X-20 (BD Biosciences) and data processed using FlowJo software (Tree Star Inc.). Dead cells were excluded from analyses using LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (cat#L23101, Life Technologies). QuantiBRITE PE calibration beads (BD Biosciences) were used to determine GD2 and CD171 antigen density on retinoblastoma cells according to the manufacturer’s instructions.
Quantibrite pe calibration beads
Manufactured by BD
QuantiBRITE PE calibration beads are a set of fluorescent beads used to calibrate the fluorescence intensity of flow cytometers. They are designed to provide a standardized reference for quantifying the expression of fluorescently-labeled proteins or other molecules on the surface of cells.
Automatically generated - may contain errors
Lab products found in correlation
Lag 3, by BD (1 mentions)
Cd171, by Miltenyi Biotec (1 mentions)
Pdcd1, by BioLegend (1 mentions)
Fortessa x20, by BD (1 mentions)
Live dead fixable green dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Biotinylated cetuximab, by Bristol-Myers Squibb (1 mentions)
Tim 3, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd11b, by BioLegend (1 mentions)
2 protocols using quantibrite pe calibration beads
1
Multiparametric Flow Cytometry Analysis of Retinoblastoma Cells
2
Quantifying GD2 Antigen on Neuroblastoma Cells
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QuantiBRITE PE calibration beads (BD Biosciences) were used to determine GD2 antigen density on neuroblastoma cells according to the manufacturer’s instructions as previously described.21 (link) To assess binding of EKTOMUN and SUREK to GD2+ neuroblastoma cell lines, we incubated 200 000 SK-N-BE(2) cells with EKTOMUN and 200 000 NXS2 cells with SUREK for 1 hour at 4°C. Subsequently, cells were stained with a PE-labeled secondary antibody directed against mouse IgG2a (cat#407108, Biolegend) for 30 min at 4°C and flow cytometric analysis was performed. To visualize trifunctional effector and target cell binding, we coincubated 2×105 NB69 cells expressing GFP_ffluc with 2×106 PBMCs with or without 1 µg/mL EKTOMUN for 5 min at 37°C. After vortexing, the mixture was fixed immediately using BD cytofix/cytoperm according to the manufacturer’s instructions. Subsequently flow cytometric analysis was performed using antibodies against CD11b (cat#101236, Biolegend), CD4 (cat#357407, Biolegend) and CD8 (cat# 300912, Biolegend). Data were analyzed using FlowJo software (BD).
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