CD4 T cells were isolated from spleen and lymph nodes by magnetic bead depletion using a CD4 T cell isolation kit (Miltenyi Biotec). Naive T cells were further isolated using naive T cell isolation kit (StemCell Technologies) or phycoerythrin (PE)-conjugated antibodies against CD25 and CD44, followed by PE magnetic bead depletion (Miltenyi Biotec). Freshly isolated naive T cells were differentiated into different T cell subsets with plate-bound αCD3 (5 µg/ml; 145-2C11) and soluble αCD28 (2 µg/ml; PV-1) in the presence of appropriate cytokines (Th0: 20 U/ml rhIL-2, 5 µg/ml αIFN-γ antibody, and 5 µg/ml αIL-4 antibody; Th1: 10 ng/ml rmIL-12 and 5 µg/ml αIL-4 antibody; Th2: 10ng/ml rmIL-4 and 5 µg/ml αIFN-γ antibody; Th17: 20 ng/ml rmIL-6, 2.5 ng/ml rhTGFβ1, 5 µg/ml αIFN-γ antibody, and 5 µg/ml αIL-4 antibody; T reg cells: 20 U/ml rhIL-2 and 5 ng/ml rhTGFβ1). All cytokines were purchased from PeproTech unless otherwise stated. For some Th17 differentiation experiments, TGF-β1, Nicotinamide (Sigma-Aldrich), or Ex-527 (Tocris) was added as indicated in the corresponding figures.
Naive t cell isolation kit
Manufactured by STEMCELL
Sourced in Canada
The Naive T cell isolation kit is a laboratory tool used to purify naive T cells from a sample. It utilizes a combination of magnetic beads and antibodies to selectively isolate the desired cell population.
Automatically generated - may contain errors
Lab products found in correlation
Human serum, by Valley Biomedical (2 mentions)
Human na ve cd8, by STEMCELL (2 mentions)
Human t activator cd3 cd28 beads, by Thermo Fisher Scientific (2 mentions)
Ex527, by Bio-Techne (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
Tgf β1, by Merck Group (1 mentions)
4 protocols using naive t cell isolation kit
1
Isolation and Differentiation of Murine T Cell Subsets
2
Isolation of Naive CD4+ T Cells
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Naive CD4+ T cells were isolated magnetically using a naive T cell isolation kit (Stemcell Technologies) according to the manufacturer's recommended protocol.
3
Generation and Expansion of CAR T Cells
4
Generation and Expansion of CAR T Cells
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CAR T cells were produced using our research-grade protocol(25 (link)) for indicated experiments. Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from the Michael Amini Transfusion Medicine Center at COH (IRB: 15283). CD8 and/or CD8 T naïve cells were isolated from PBMCs with Human Naïve CD8+ or Naive T Cell Isolation Kits, respectively (Stemcell Technologies, Vancouver, Canada). T cells were cultured in vivo-15 (Lonza, Basel, Switzerland) supplemented with 10% human serum (Valley biomedical, Winchester, Virginia, USA) and 100U/mL human IL-2, and activated with Human T-Activator CD3/CD28 beads (Life Technologies, Carlsbad, California, USA) for 24 hours before transduction with lentivirus at MOI 1. Cultures were maintained at 0.5–1×106 cells/mL in medium containing 100 U/mL IL-2. After 7 days, CD3/CD28 beads were removed by Dynamag-2. GFP-positive single CAR T cells or EGFR-positive dual CAR T cells were enriched by FACS, activated and expanded with CD3/CD28 bead stimulation for another 7 days. CD3/CD28 beads were removed by Dynamag-2 before use. T cells from each donor were used to produce un-transduced T-cell controls (mock) in parallel to CAR T cells.
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