Easysep human t cell enrichment kit

Manufactured by STEMCELL

Sourced in Canada

The EasySep Human T Cell Enrichment Kit is a laboratory product designed to isolate T cells from human samples. It utilizes magnetic particles and a specialized buffer system to selectively enrich for T cells, allowing for their subsequent analysis or use in downstream applications.

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EasySep kits (21 citations) EasySep Human CD4+ T cells Enrichment Kit (3 citations)

52 protocols using easysep human t cell enrichment kit

Profiling MGAT5 Expression in UC

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CD3+ T cells were isolated from 24 blood samples from patients with UC (18 with active disease—with Mayo endoscopic subscore > 0). A density gradient centrifugation (with Lymphoprep, STEMCELL) was used to obtain a peripheral blood mononuclear cells suspension, and CD3+ cells were magnetically sorted using the EasySep Human T Cell Enrichment Kit (STEMCELL) following the manufacturer's instructions. CD3+ T cells were also isolated from 14 fresh colon biopsies (12 with active disease), through mechanical dissociation (36 (link)) and posterior magnetically sorted with EasySep Human T Cell Enrichment Kit (STEMCELL).
RNA was isolated using RNAqueous-Mirco kit (Ambion), and MGAT5 and GAPDH (used as reference genes) expression levels were detected using the Hs00159136_m1 and Hs02758991_g1 probes, respectively, by TaqMan Real-time PCR as previously described (33 (link)). The MGAT5 expression levels were evaluated in patients who display different genetic variants of the SNPs rs1257220, rs3814022, and rs4953911 and were normalized to GAPDH housekeeping expression by delta-CT method, and the results are shown as relative expression.

Pereira M.S., Durães C., Catarino T.A., Costa J.L., Cleynen I., Novokmet M., Krištić J., Štambuk J., Conceição-Neto N., Machado J.C., Marcos-Pinto R., Magro F., Vermeire S., Lauc G., Lago P, & Pinho S.S. (2020). Genetic Variants of the MGAT5 Gene Are Functionally Implicated in the Modulation of T Cells Glycosylation and Plasma IgG Glycome Composition in Ulcerative Colitis. Clinical and Translational Gastroenterology, 11(4), e00166.

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Isolation and Enrichment of Human T Cells

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Human PBMCs were isolated from the buffy coat of heparinized whole blood after Ficoll-Hypaque (Histopaque-1077; Sigma-Aldrich) density-gradient centrifugation. The PBMCs were washed twice with Dulbecco’s phosphate-buffered saline (DPBS), counted, and resuspended in 2% FBS-1 mM EDTA-DPBS (sorting medium) at 5 × 10⁷ cells/mL. T cells were isolated from the PBMCs by magnetic-based negative selection using the EasySep^TM human T cell enrichment kit (Stemcell technologies) according to the manufacturer’s protocol. The purity of the T cells (>95%) was checked by staining with anti-CD3-APC/Cy7 (clone SK7, Biolegend). The T cells were then resuspended in 10% FBS-AIM-V medium (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA).

Mahasongkram K., Glab-ampai K., Kaewchim K., Saenlom T., Chulanetra M., Sookrung N., Nathalang O, & Chaicumpa W. (2023). Agonistic Bivalent Human scFvs-Fcγ Fusion Antibodies to OX40 Ectodomain Enhance T Cell Activities against Cancer. Vaccines, 11(12), 1826.

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Activated T Cell Transduction for CAR T Therapy

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According to the product instructions, we selected human CD3⁺ T cells with the EasySepTM Human T Cell Enrichment Kit (Stem Cell Technologies). T cells were first activated using ImmunoCult™ Human CD3/CD28 T Cell Activator (Stem Cell Technologies), then CAR T cells were prepared by transducing lentivirus into activated T cells and cultured in T cell medium containing 100 ng/ml IL-2. The CAR T cells were used for the subsequent in vivo and in vitro experiments on the 10th or 11th day after activation if cell viability was greater than 90%.

Li C., Zuo S., Shan L., Huang H., Cui H, & Feng X. (2024). Myeloid leukemia-derived galectin-1 downregulates CAR expression to hinder cytotoxicity of CAR T cells. Journal of Translational Medicine, 22, 32.

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T Cell Activation Protocol

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T cells were enriched using the EasySep human T cell enrichment kit (cat No. 19051, STEMCELL Technologies, Vancouver, Canada). After T cell enrichment, the cells were suspended in complete medium (RPMI 1640, 50%, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing NEAA, HEPES, L-GlutaMax, 2-mercaptoethanol, FBS, and antibiotics. Brefeldin A was added to all groups in order to lock the produced transcripts and proteins inside the cells. Interleukin (IL)-2 was added to all groups to ensure T cell survival during the incubation period. All donor samples were divided into three activation groups: group 1, resting control; group 2, 50 ng/mL PMA (cat No. P8139, 1 mg, Sigma Aldrich, St. Louis, MO, USA) and 1.34 µM ionomycin (cat No. I0634, 1 mg, Sigma) for chemical stimulation and group 3, Dynabeads human T-activator CD3/CD28 (cat No. 11131D, Gibco, Thermo Fisher Scientific) added at a 1:1 ratio (beads: cells) for physiological activation. Each group was incubated for 4 h at 37.5℃ in a 5% CO₂ incubator. After 4 h of incubation, the cells were harvested and used in further experiments.

Lee J.H., Lee B.H., Jeong S., Joh C.S., Nam H.J., Choi H.S., Sserwadda H., Oh J.W., Park C.G., Jin S.P, & Kim H.J. (2023). Single-cell RNA sequencing identifies distinct transcriptomic signatures between PMA/ionomycin- and αCD3/αCD28-activated primary human T cells. Genomics & Informatics, 21(2), e18.

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Allostimulation Assay with MDDC-Primed T Cells

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Allostimulation assays were performed by culturing together 1h-MV pulsed, 24 h-resting MDDCs with allogeneic CFSE-labelled T cells. T cells were isolated from healthy donors' PBMCs by negative magnetic selection using the Easysep Human T cell Enrichment Kit (Stemcell Technologies), and stained with CFSE (0.4 M; Invitrogen).
iDCs were co-cultured with T cells at different ratios, from 1:20 (5000 MDDCs: 100,000 T cells) to 1:160 (625 MDDCs: 100,000 T cells). As a positive control, T cells were stimulated with Phorbol 12-Myristate 13-Acetate (PMA, 0.6 ng/ml, Sigma Aldrich) and Ionomycin calcium salt (200 ng/ml, Sigma Aldrich).
After 4.5 days of culture, proliferation was assessed by flow cytometry (LSR Fortessa Analyzer, BD). Proliferative T cells were gated by diluted CFSE intensity.

Evans-Osses I., Mojoli A., Monguió-Tortajada M., Marcilla A., Aran V., Amorim M., Inal J., Borràs F.E, & Ramirez M.I. (2017). Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro. European journal of cell biology, 96(2).

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CD3+ T-cell Enrichment Protocol

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The CD3+ T-cells were further isolated using the EasySep™ Human T Cell Enrichment Kit (Stem Cell technologies, Vancouver, BC, Canada). T-cell suspension was prepared in phosphate-buffered saline (PBS) within a 5 mL polystyrene tube (Becton Dickinson (BD), Franklin Lakes, NJ, USA) at a density of 5 × 10⁷ cells/mL. EasySep™ Human T Cell Enrichment Cocktail and EasySep™ D Magnetic Particles was added at 50 μL/mL cells, and the cells were incubated and placed into a magnet for cell separation according to the manufacturer’s protocol. The negatively selected CD3+ T-cell population were cultured.

Munisvaradass R., Kumar S., Govindasamy C., Alnumair K.S, & Mok P.L. (2017). Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells. International Journal of Molecular Sciences, 18(9), 1797.

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Isolation and Expansion of Human T Cells

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Example 1

The mononuclear cells were isolated from donor blood, then density gradient centrifugation was performed with Histopaque-1077 (Sigma-Aldrich), and the T cells (EasySep Human T Cell Enrichment Kit, Stemcell Technologies) were enriched. The T cells were activated, cultured and expanded using coupled anti-CD3/anti-CD28 magnetic beads; X-vivol5 (300 IU/ml rhIL2) was used as the medium; and then all cells were cultured in a constant temperature incubator at 37° C., 5% CO₂.

US11840575B2. Engineered immune cells targeting BCMA and their uses thereof (2023-12-12). GRACELL BIOTECHNOLOGIES (SHANGHAI) CO., LTD. [CN]. Inventors: Hua Zhang [CN], Huan Shi [CN], Lianjun Shen [CN], Wei Cao [CN], Liping Liu [CN].

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PBMC and T-cell Isolation for Functional Analyses

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Peripheral blood mononuclear cells (PBMCs) and CD3⁺ T cells were isolated from whole blood as previously described (22 (link)). Briefly, PBMCs were isolated from centrifugation of whole blood using Ficoll-Paque (GE Healthcare Bio-sciences) density gradient and CD3⁺ cells were further isolated from the PBMCs by negative selection using EasySep™ Human T-cell enrichment kit (StemCell Technologies). T cells were maintained in RPMI-1640 medium supplemented with 10% human serum, 200 u/ml penicillin, 200 mg/ml streptomycin, 1 mM L-glutamine, 25 mM HEPES (Sigma-Aldrich) and cells were used immediately for calcium measurement and electrophysiology.

Chimote A.A., Hajdu P., Sfyris A.M., Gleich B.N., Wise-Draper T., Casper K.A, & Conforti L. (2016). Kv1.3 channels mark functionally competent CD8+ tumor infiltrating lymphocytes in head and neck cancer. Cancer research, 77(1), 53-61.

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T Cell Receptor Sequencing from PBMCs

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PBMCs from Donors A and B were isolated by Ficoll-Paque (GE Healthcare) gradient centrifugation and cryopreserved. T cells were purified from thawed PBMCs using the EasySep Human T cell enrichment kit (STEMCELL Technologies). RNA was extracted from purified human T cells using the RNeasy Plus Minikit (Qiagen). 0.5 μg Donor B RNA was mixed with 4.5 μg Donor A RNA to create sample C for quantitation experiments. 4 μg each of Donor A, Donor B, and sample C RNA was reverse transcribed using a TCRB chain constant region reverse primer with Superscript III First-Strand Synthesis System (Invitrogen). cDNA was column purified with the Oligo Clean and Concentrator Kit (Zymo Research). 100 ng of purified cDNA from each sample was sent to Adaptive Biotechnologies for the immunoSEQ hsTCRb Deep sequencing service or used for FR3AK-seq PCR. For the ArcherDx Immunoverse analysis, RNA was extracted from matched Donor A and B PBMCs and mixed at the same 9:1 ratio to create sample C. 800 ng of RNA from each sample was input into the Immunoverse assay. The concordance between FR3AK-seq and Immunoverse remained high despite the use of PBMC RNA rather than T cell RNA in the Immunoverse assay. Primer sequences are provided in Table S1.

Montagne J.M., Zheng X.A., Pinal-Fernandez I., Milisenda J.C., Christopher-Stine L., Lloyd T.E., Mammen A.L, & Larman H.B. (2020). Ultra-efficient sequencing of T Cell receptor repertoires reveals shared responses in muscle from patients with Myositis. EBioMedicine, 59, 102972.

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Quantifying Treg-mediated Suppression of T-cell Proliferation

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Monocyte-derived dendritic cells (moDCs) were generated by plating PBMCs on 100-mm culture dishes (BD Biosciences) for 1 h at 378C/5% CO 2 in AIM-V medium (Life Technologies) with 2.5% heat-inactivated human serum (Sigma Aldrich), washing away nonadherent cells and culturing adherent cells with 1000 IU/mL IL-4 and 1000 IU/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; both Miltenyi Biotec) for 4 days. Responder T cells were isolated from PBMCs using EasySep Human T-Cell Enrichment Kit (StemCell Technologies), stained with PKH26 Red (Sigma Aldrich) or Cell Proliferation Dye eFluor450 (eBioscience) and stimulated with a-CD3/CD28 Dynabeads (Life Technologies) at a 10:1 ratio (cells:beads) for 4 days or allogeneic moDCs at a 2:1 ratio (T cells:moDCs) for 6 days. Tregs were added at a Treg:responder cell ratio of 1:2.5 to 1:20. After culture, supernatants were collected and cells were stained with a-human CD3, CD4, and CD8 mAb (Table S1). Data were acquired and analyzed as described above. Suppressive capacity of Tregs was defined by calculating percentage inhibition of proliferation based on the Division Index of CD8 þ responder cells.

Dijke I.E., Hoeppli R.E., Ellis T., Pearcey J., Huang Q., McMurchy A.N., Boer K., Peeters A.M., Aubert G., Larsen I., Ross D.B., Rebeyka I., Campbell A., Baan C.C., Levings M.K, & West L.J. (2016). Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(1).

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