Previously frozen PBMCs were incubated with the cell-permeant dye indo-1, AM (Molecular Probes) for 45 min at 37°C and then surface-stained with anti–CD8 PE/Cy5 (BioLegend), anti–CD27-APC (BD Biosciences), anti–CD45RO-PE (BD Biosciences), anti–CD45RA-FITC (BioLegend), anti–CD4-PerCP/Cy5.5 (BioLegend), and anti–CD19-PE/Cy7 (BioLegend). The indo-1 fluorescence ratio was acquired as a function of time on an LSR II flow cytometer, and kinetics curves were generated using the FlowJo software. Collection of a 30-s or 1-min baseline was followed by stimulation with 10 μg/mL soluble anti-CD3 (clone UCHT1, BioLegend), 20 μg/mL anti-κ + anti-λ F(ab’)2 (SouthernBiotech), or 1 μg/mL ionomycin. All the calcium flux profiles were generated using a standard protocol.
Soluble anti cd3 clone ucht1
Manufactured by BioLegend
Soluble anti-CD3 (clone UCHT1) is an antibody product that binds to the CD3 receptor on T cells. Its core function is to activate T cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Indo 1 am, by Thermo Fisher Scientific (1 mentions)
Anti cd19 pe cy7, by BioLegend (1 mentions)
Anti cd45ro pe, by BD (1 mentions)
Anti cd4 percp cy5, by BioLegend (1 mentions)
Anti cd27 apc, by BD (1 mentions)
Anti cd45ra fitc, by BioLegend (1 mentions)
Anti cd8 pecy5, by BioLegend (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Ficol, by Eurobio Scientific (1 mentions)
2 protocols using soluble anti cd3 clone ucht1
1
Calcium Flux Profiling of T and B Cells
2
Isolation and Activation of CD4+ T Cells
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Healthy donor blood prepared as a buffy coat was obtained from Etablissement Français du Sang (EFS) (agreement with Institut Pasteur C CPSL UNT, 15/EFS/023). Blood was overlayed on Ficol (EuroBio) at a ratio of 2:1 v/v blood to Ficol and centrifuged at 1,800 rpm for 30 minutes at a minimum acceleration/deceleration to obtain peripheral blood mononuclear cells (PBMCs). CD4+ T cells were then purified from PBMCs by negative selection using StemCell EasySep TM Human CD4+ T cell Isolation Kit. Cells were counted and cultured in RPMI-1640 containing Glutamax (ThermoFisher), 10% fetal bovine serum (FBS), penicillin-streptomycin (ThermoFisher) (100 U/ml) and IL-2 (Myltenyi) (100U/ml) (thereafter referred to as culture medium) at 10 6 cells/ml in 37° degree, 5% CO 2 humidified incubator. Cells were activated with soluble anti-CD3 (clone UCHT-1) (Biolegend) for 5 days prior to infection or analysis as previously described (27) .
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