Primary human T cells were procured and cultured as described previously (41 (link)). Briefly, peripheral blood mononuclear cells were collected from anonymous healthy donors by aphaeresis. T cells were isolated from these aphaeresis products by the University of Pennsylvania Human Immunology Core using Lymphoprep and RosetteSep Human T Cell Enrichment Cocktail kits (STEMCELL Technologies) according to the manufacturer’s instructions. Primary human T cells were cultured at 37°C in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, 10 mM Hepes, penicillin (100 U/ml), and streptomycin (100 μg/ml). Nalm6 cells (American Type Culture Collection) were cultured at 37°C in the same medium that was used for T cells and refed every other day to a concentration of 0.25 × 106 cells/ml. Immediately before being used as stimulator cells, Nalm6 cells were irradiated with 100 Gy using an x-ray irradiator.
Rosettesep human t cell enrichment cocktail kit
Manufactured by STEMCELL
Sourced in Canada
The RosetteSep Human T Cell Enrichment Cocktail kits are designed to isolate T cells from human whole blood or buffy coat samples. The cocktail contains antibody complexes that crosslink unwanted cells to red blood cells, allowing the T cells to be easily separated by density centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Nalm6 cells, by American Type Culture Collection (1 mentions)
Hyaluronidase, by Yeasen (1 mentions)
Pha l, by Merck Group (1 mentions)
Collagenase 1, by Yeasen (1 mentions)
Collagenase 4, by Yeasen (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
4 protocols using rosettesep human t cell enrichment cocktail kit
1
Primary Human T Cell Isolation and Culture
2
Single-Cell Isolation and T-Cell Activation
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All the experiments involving human samples were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards, based on informed consent. Tumor tissues were obtained from a patient with HCC who underwent surgery at Fudan University Shanghai Cancer Center. The samples were enzymatically digested using collagenase I, collagenase IV, and hyaluronidase (YEASEN, Shanghai, China) for 1 hr at 37°C. Single-cell suspensions were filtered with 70-µm filter units (Coring Falcon, NY, USA), and then incubated with diabody-mp or diabody-pm at 37°C for 48 hr. The hPBMCs were isolated from healthy individuals using Ficoll-Paque (GE Healthcare, Little Chalfont, UK). Autologous T cells were enriched using RosetteSep™ human T cell enrichment cocktail kits (STEMCELL technology, Canada). The hPBMCs were activated with PHA-L (2 µg/mL) (Sigma-Aldrich, MO, USA), and T cells were activated with 100 ng/mL of anti-human CD3 and anti-human CD28 antibodies (PeproTech, NJ, USA) for 48 hr.
3
T Cell Genomic Analysis in RRMS Patients
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We recruited 23 relapsing remitting MS (RRMS) patients and 12 age and gender-matched healthy controls for this study. Patients were enrolled at the Multiple Sclerosis Unit from the Complejo Hospitalario de Navarra and controls were recruited among hospital workers or patient’s next of kin. All RRMS patients fulfilled the revised McDonald criteria [17 (link)] and none of the participants was under vitamin D supplementation when blood samples were collected.
For all 35 subjects T cells were isolated from whole blood by negative selection with RosetteSep™ Human T Cell Enrichment Cocktail kit (StemCell Technologies Inc, Vancouver, BC, Canada). Next, genomic DNA was extracted by standardized methods [18 (link)] and stored at -20°C until further use.
The study was approved by the Ethics Committee of the Complejo Hospitalario de Navarra for using human subjects and written informed consent was obtained from all subjects participating in the study.
For all 35 subjects T cells were isolated from whole blood by negative selection with RosetteSep™ Human T Cell Enrichment Cocktail kit (StemCell Technologies Inc, Vancouver, BC, Canada). Next, genomic DNA was extracted by standardized methods [18 (link)] and stored at -20°C until further use.
The study was approved by the Ethics Committee of the Complejo Hospitalario de Navarra for using human subjects and written informed consent was obtained from all subjects participating in the study.
4
Isolation and Purification of Human CD3+ Cells
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Human CD3+ cells were isolated from 8 mL of heparinized whole blood by negative selection using the EasySep Human T Cell Isolation Kit (#17951, Stemcell Technologies, Vancouver, BC, Canada) in 2017, and the RosetteSep Human T Cell Enrichment Cocktail Kit (#15021, Stemcell Technologies) in 2021. Purification was confirmed by flow cytometry, obtaining >95% purity in both cases. CD3+ pellets were stored at -80°C until use.
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