Rosettesep human t cell enrichment cocktail

Manufactured by STEMCELL

Sourced in Canada, United States

The RosetteSep Human T Cell Enrichment Cocktail is a product designed for the isolation of human T cells from whole blood or buffy coat samples. It utilizes an antibody cocktail that binds to non-T cells, allowing for the negative selection of the desired T cell population.

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RosetteSep (157 citations)

59 protocols using rosettesep human t cell enrichment cocktail

Isolation of CD3+ Lymphocytes from Patient Blood

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Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of patients prior to lymphodepletion by density gradient with Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ, USA). CD3⁺ untouched lymphocytes were separated from the peripheral blood of patients before lymphodepletion by RosetteSep^TM Human T Cell Enrichment Cocktail (StemCell Technologies, Vancouver, Canada) using negative selection. Briefly, 4 mL of whole blood was mixed with 200 µL RosetteSep™ Cocktail and centrifuged over a density gradient medium (Ficoll-Hypaque, Pharmacia Biotech, Piscataway, NJ, USA). CD3⁺ enriched population at the interface between the plasma and the density gradient medium was collected.

Beider K., Itzhaki O., Schachter J., Grushchenko-Polaq A.H., Voevoda-Dimenshtein V., Rosenberg E., Ostrovsky O., Devillers O., Shapira Frommer R., Zeltzer L.A., Toren A., Jacoby E., Shimoni A., Avigdor A., Nagler A, & Besser M.J. (2022). Molecular and Functional Signatures Associated with CAR T Cell Exhaustion and Impaired Clinical Response in Patients with B Cell Malignancies. Cells, 11(7), 1140.

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Isolation of NK and T Cells from Human Blood

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Blood samples from healthy donors were collected by the local institute of Experimental Haematology and Transfusion medicine (IHT) at the University Hospital of Bonn. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll–Hypaque density gradient centrifugation (specific gravity, 1.077 g/mL; Lympholyte^TM, Cedarlane, Burlington, Canada). NK cells were negatively selected from PBMCs using the untouched NK cell isolation kit (human) onto LD columns (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Purity of enriched NK cells was assessed by flow cytometry. For isolation of T cells, 10 mL of Buffy coat was incubated with 500 µL RosetteSep^TM Human T Cell Enrichment Cocktail (Stemcell Technologies, Vancouver, Canada) and proceeded according to the manufacturer’s instructions.

Schönberger S., Kraft D., Nettersheim D., Schorle H., Casati A., Craveiro R.B., Mohseni M.M., Calaminus G, & Dilloo D. (2020). Targeting EpCAM by a Bispecific Trifunctional Antibody Exerts Profound Cytotoxic Efficacy in Germ Cell Tumor Cell Lines. Cancers, 12(5), 1279.

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T Cell Activation and Cytokine Secretion

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Human T cells were isolated from the blood of healthy adult volunteers using RosetteSepTM Human T Cell Enrichment Cocktail (STEMCELL, #15061). Isolated human T cells (1 × 10⁵ cells) were inoculated onto 96-well Nunc MaxiSorp^™ plates precoated with anti-CD3 antibody (OKT3, Biolegend, 1 μg/mL) and screened anti-CD137 antibodies. After 3 days of incubation, secreted IL-2 and IFN-γ levels were determined by ELISA assay.

Huang P.L., Kan H.T., Hsu C.H., Hsieh H.T., Cheng W.C., Huang R.Y, & You J.J. (2023). A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity. Journal of Translational Medicine, 21, 346.

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Isolation of T Lymphocytes from Blood

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EDTA or heparin anticoagulant peripheral blood (10 mL) was collected from the patients and controls as previous described [11 (link),13 (link)]. First, we obtained the lymphocytes by inverted-culture mononuclear leucocytes, which were purified by lymphocyte separation medium (TBD Biological Manufacture Co., Ltd., Tianjin, China). Then, the T lymphocytes were purified by negative selection using the RosetteSep^TM human T cell enrichment cocktail (15021, Stem Cell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. The cells were then incubated in the RPMI-1640 medium at 37 °C.

Zeng J.Y., Du J.J., Pan Y., Wu J., Bi H.L., Cui B.H., Zhai T.Y., Sun Y, & Sun Y.H. (2016). Calcium-Sensing Receptor in Human Peripheral Blood T Lymphocytes Is Involved in the AMI Onset and Progression through the NF-κB Signaling Pathway. International Journal of Molecular Sciences, 17(9), 1397.

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Isolation and Manipulation of Cell Lines

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Target cells used were the luciferase transduced PC-3 (human prostate cancer), the UC-3 (human bladder cancer) cell lines, MyLa-2059 (human T-cell non-Hodgkin lymphoma), 4T1 (murine breast cancer), and U2OS (Human Bone Osteosarcoma cells). Cell lines were kindly provided by our collaborators at Vancouver Prostate Centre, British Columbia, Canada and Clausen group, Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark.
Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood from healthy human donors by density gradient medium (LymphoprepTM). When isolating T cells from whole blood, the blood was incubated with RosetteSepTM Human T cell Enrichment Cocktail (Stemcell, 15021) according to manufactures instructions. Depletion of T cells from isolated PBMCs were performed using RosetteSepTM Human CD3 Depletion Cocktail.

Nordmaj M.A., Roberts M.E., Sachse E.S., Dagil R., Andersen A.P., Skeltved N., Grunddal K.V., Erdoğan S.M., Choudhary S., Gustsavsson T., Ørum-Madsen M.S., Moskalev I., Tian W., Yang Z., Clausen T.M., Theander T.G., Daugaard M., Nielsen M.A, & Salanti A. (2021). Development of a bispecific immune engager using a recombinant malaria protein. Cell Death & Disease, 12(4), 353.

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Purification and Analysis of MAIT Cells

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Whole blood from ten healthy individuals was obtained from the Red Cross Blood Center. The study protocol was approved by the Institutional Review Board of the Red Cross. For the cell sorting of MAIT cells, CD161⁻ conventional T cells, TCR_α7.2+ CD161− T cells and total T cells were isolated from the whole blood to >95% purity. This was achieved through negative selection using a RosetteSep Human T Cell Enrichment Cocktail (StemCell Technologies, Vancouver, British Columbia, Canada) and incubating with anti-CD3-APC-Cy7, anti-TCRα7.2-FITC, and anti-CCR161-PE-Cy7 in HBSS supplemented with 4% fetal bovine serum for 15 min. The cells were washed, resuspended in Hank’s Balanced Sal Solution, and sorted into a MAIT cell fraction. Cell subsets were isolated to nearly 100% purity using the FACSARIA III cytometer (BD Biosciences, San Jose, CA, USA.) Cell subsets from three different donors were used for RNA sequencing.

Park D., Kim H.G., Kim M., Park T., Ha H.H., Lee D.H., Park K.S., Park S.J., Lim H.J, & Lee C.H. (2019). Differences in the molecular signatures of mucosal-associated invariant T cells and conventional T cells. Scientific Reports, 9, 7094.

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T cell stimulation with co-inhibitory signals

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Peripheral blood was purchased from New York blood center. Total CD3⁺ T cells were isolated by density gradient centrifugation (Lymphoprep) and negative selection using the RosetteSep human T cell enrichment cocktail (Stem Cell). Primary T cells were directly used in stimulation assays. In vitro T cell cultures were maintained in complete RPMI, containing 10% FBS, MEM nonessential amino acids, 1mM sodium pyruvate, 100 IU/ml of penicillin, 100 μg/ml streptomycin and GlutaMAX-I. Jurkat T cells were obtained from the American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. MC38 cells were provided by Kerafast and maintained in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. HEK 293T cells were obtained from the American Type Culture Collection and maintained in 5% CO₂ at 37°C in DMEM media supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. Cells were stimulated with magnetic beads (ratio of 1:5 cells per bead), which were conjugated with the following protein combinations (the ratio in parentheses indicates the relative concentration of each protein): anti-CD3/IgG1 (1:3), anti-CD3/PDL2-Fc/IgG1 (1:2:1), anti-CD3/anti-CD28/IgG1 (1:1:2), or anti-CD3/anti-CD28/PD-L2-Fc (1:1:2).

Peled M., Tocheva A.S., Adam K, & Mor A. (2021). VRK2 inhibition synergizes with PD-1 blockade to improve T cell responses. Immunology letters, 233, 42-47.

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Isolation and Differentiation of Primary Immune Cells

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Human primary T cells and monocytes were isolated from healthy subjects’ peripheral blood by using RosetteSep human T cell enrichment cocktail and RosetteSep human monocyte enrichment cocktail (Stemcell technologies, Vancouver, BC, Canada), followed by Ficoll Paque gradient centrifugation. Macrophages were further derived from monocytes via replacing half of the culture medium each day for 7 days.

Yang C.A., Li J.P., Yen J.C., Lai I.L., Ho Y.C., Chen Y.C., Lan J.L, & Chang J.G. (2018). lncRNA NTT/PBOV1 Axis Promotes Monocyte Differentiation and Is Elevated in Rheumatoid Arthritis. International Journal of Molecular Sciences, 19(9), 2806.

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Expansion and Transduction of Human T Cells

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Blood collected from three healthy donors was used to isolated T cells respectively using the RosetteSep™ Human T Cell Enrichment Cocktail (STEMCELL, Canada) and the T cells were cultured in RPMI 1640 medium (Life Technologies, USA) containing 10% FBS (Life Technologies, USA) with 200 U/ml IL-2 (PeproTech, USA). T cells were activated by CD3/CD28 Dynabeads (Life Technologies, USA) according to the manufacturer’s instructions. After 48 h, lentiviral particles were added to the cultures at a multiplicity of infection (MOI) of 10 in the presence of polybrene at a final concentration of 8 μg/ml. The CAR-T cells were counted on alternate days and fresh medium was added to the cultures to maintain the cell density at 1 × 10⁶ cells/ml. Four days after T cells were infected with CAR lentivirus, total T cells were used for in vitro experiments. When the T cells were expanded in vitro for an additional 6 days, the cells were collected and used for the in vitro experiments.

Yang D., Sun B., Dai H., Li W., Shi L., Zhang P., Li S, & Zhao X. (2019). T cells expressing NKG2D chimeric antigen receptors efficiently eliminate glioblastoma and cancer stem cells. Journal for Immunotherapy of Cancer, 7, 171.

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Isolation of Human T Lymphocytes

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T lymphocytes were isolated from buffy-coats of a healthy volunteer, using RosetteSep^® Human T Cell Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada) according to manufacture protocol. The enriched T cells were washed two times with phosphate buffer saline (PBS) +2% fetal bovine serum. The lymphocytes were suspended in completed Roswell Park Memorial Institute (RPMI1640) (RPMI1640 with L-glutamine (Sigma) supplemented with 10% fetal calf serum (FCS) (Gibco) and 1% penicillin/streptomycin (Roche)) and T cell suspension was adjusted at 2 × 10⁶ Cells/ml.

Alipour R., Adib M., Hashemi-Beni B, & Sadeghi F. (2014). The effect of stem cell from human exfoliated deciduous teeth on T lymphocyte proliferation. Advanced Biomedical Research, 3, 202.

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