Human PBMCs were isolated using Ficoll-Hypaque (GE-Healthcare; GE17–1440-02) gradient separation. For RNA-seq CD4+ T cells were enriched using CD4 microbeads (Miltenyi; 130–045-101) and 4×106 cells/ml were resuspended in RPMIc with 50 U/ml IL-2 (Immunotools; 11340023) in 24-well plate wells and stimulated for 20h with 25µl/ml ImmunoCult CD3/CD28 T cell activator (Stemcell; 10971) prior to staining and cell sorting.
Immunocult cd3 cd28 t cell activator
Manufactured by STEMCELL
Sourced in United States
Immunocult CD3/CD28 T cell activator is a product designed to activate human T cells in vitro. It comprises of immobilized anti-CD3 and anti-CD28 antibodies that mimic the natural activation signals received by T cells, leading to their proliferation and differentiation.
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Lab products found in correlation
Countess 2, by Thermo Fisher Scientific (3 mentions)
Recombinant murine il 2, by BioLegend (2 mentions)
Recombinant murine il 4, by BioLegend (2 mentions)
Easysep human cd4 cd127lowcd25 regulatory t cell isolation kit, by STEMCELL (2 mentions)
Human il 2, by STEMCELL (2 mentions)
Pma ionomycin, by BioLegend (2 mentions)
Easysep mouse cd4 cd25 regulatory t cell isolation kit, by STEMCELL (2 mentions)
Immunocult xf t cell expansion medium, by STEMCELL (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Cd4 microbeads, by Miltenyi Biotec (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Interleukin 2 (il 2), by Immunotools (1 mentions)
Interleukin il 2, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Caisson (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin, by Caisson (1 mentions)
4 protocols using immunocult cd3 cd28 t cell activator
1
CD4+ T Cell Activation and Isolation
2
Crbn Knockout Regulatory T Cell Culture
3
Crbn Knockout Regulatory T Cell Culture
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FLP293T derived stable cell lines were cultured in DMEM supplemented with 10% dialyzed fetal bovine serum (FBS). Wildtype and Crbn−/− Jurkat cells were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin in a 37°C incubator with 5% CO2. CD4+ CD25+ regulatory T cells were purified from spleens from wildtype or CrbnI391V/I391V mice using the EasySep Mouse CD4+ CD25+ Regulatory T cell Isolation Kit (StemCell Technologies). T cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 2 mM GlutaMax, 10 mM HEPES, and 50 μM 2-mercaptoethanol, as well as 5 ng/ml recombinant murine IL-2 (Biolegend) and 20 ng/ml recombinant murine IL-4 (Biolegend). Human regulatory T cells were isolated from peripheral blood mononuclear cells using the EasySep Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (StemCell Technologies) and expanded for 12–14 days in the presence of 500 U/ml human IL-2, Immunocult CD3/CD28 T cell activator (StemCell Technologies), and compound in ImmunoCult-XF T Cell Expansion Medium (StemCell Technologies). Cells were stimulated with 1X PMA/ionomycin (Biolegend) for 1h followed by the addition of brefeldin A (Biolegend) for another 3h.
4
Expansion and Activation of Isolated T Cells
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Isolated T cells were suspended at 106 cells/mL in RPMI 1640 media with L‐glutamine (Caisson Labs, Smithfield, UT, USA) supplemented with 10% fetal bovine serum (FBS; Sigma‐Aldrich), 1% (v/v) of penicillin/streptomycin solution, 2.5 μg/mL amphotericin B (Caisson Labs), 25 mM HEPES (Caisson Labs), 100 ng/mL Interleukin (IL)‐2 (PeproTech, Cranbury, NJ), and 25 μL/mL ImmunoCult™ CD3/CD28 T cell activator (StemCell Technologies). Cells were seeded (106 cells/well) in a non‐treated, flat‐bottom, 24‐well tissue culture plate (CellTreat, Pepperell, MA, USA) and incubated at 37°C with 5% CO2 for 3 days. On day 3, cells were collected from the plate, spun down by centrifugation at 250 × g for 10 min, re‐suspended in culture media without activator (0.25 × 106 cells/mL), and seeded in a new 24‐well plate (0.25 × 106 cells/well). Culture media supplemented with 100 ng/mL of IL‐2 was replaced every 3 days. When the total cell count for the sample reached 107, the cells were re‐suspended at a concentration of 106 cells/mL and culture continued in a 250 mL suspension culture flask (CellTreat, Pepperell, MA, USA). Total numbers of viable cells were determined using an automated cell counter with Trypan Blue (Countess II, ThermoFisher, Waltham, MA) before each media exchange.
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