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Cd4 t cell isolation kit

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The CD4+ T cell isolation kit is a laboratory tool designed to isolate CD4+ T cells from a mixed cell population. It utilizes magnetic bead-based technology to selectively separate CD4+ T cells from other cell types present in the sample.

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Lab products found in correlation

Lymphoprep, by STEMCELL (4 mentions) Facsaria, by BD (4 mentions) Ficoll paque plus, by GE Healthcare (4 mentions) 96 well round bottom plate, by Corning (3 mentions) Human ab serum, by Gemini Bio (3 mentions) Gm csf, by Thermo Fisher Scientific (2 mentions) Anti human cd3 ucht1, by BD (2 mentions) Soluble anti human cd28 28.2, by BD (2 mentions) Recombinant human il 12, by R&D Systems (2 mentions) Sgk1 inhibitor gsk650394, by Bio-Techne (2 mentions) Akt inhibitor mk2206, by Bio-Techne (2 mentions) Frizzled 8 fc chimera protein, by R&D Systems (2 mentions) Pkf115 584, by Bio-Techne (2 mentions) Human serum, by Gemini Bio (2 mentions) Nitroblue tetrazolium chloride, by Merck Group (2 mentions) Multiscreen ip filter plate, by Merck Group (2 mentions) Cfa h37 ra, by BD (2 mentions) Apob peptide, by Merck Group (2 mentions) Anti ifn γ, by Merck Group (2 mentions) 5 bromo 4 chloro 3 indol phosphate p toluidine salt, by Merck Group (2 mentions)

Close spelling variants of this product

Isolation kits (7 citations) Cell isolation kits (4 citations) CD4+ T cells isolation kit (3 citations)

35 protocols using cd4 t cell isolation kit

1

Generation and Maintenance of Th1 and Th2 Cell Lines

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Mouse Th1 and Th2 cell lines were generated from MBP TCR-Tg mice (46 (link)) as described previously (10 (link)). Th cell lines were not transformed and, therefore, were maintained by stimulation with MBPAc1−11 and irradiated splenocytes in the presence of recombinant human (rh) IL-2 (Miltenyi) every 7–10 days. T cells collected 7–10 days after activation with MBPAc1−11 and irradiated splenocytes provided the resting Th cell condition. To avoid the presence of non-T cells in in vitro experiments, resting Th1 or Th2 cell lines were activated with anti-CD3/CD28 for the indicated lengths of time. Mouse naive CD4+ T cells were isolated from spleens and lymph nodes using the mouse naive CD4+ T cell isolation kit (Miltenyi or STEMCELL Technologies) and activated using 5 μg/ml coated anti-CD3 and 2 μg/ml soluble CD28. Human Th1 and Th2 cells were generated by isolating CD4+ T cells with a CD4+ T Cell Isolation Kit (STEMCELL Technologies) from human whole blood leukocytes from normal donors and activating on anti-CD3/CD28 Dynabeads (Thermo Fisher) under Th1 (rhIL-12 + anti-hIL-4) or Th2 (rhIL-4 + anti-hIL-12 + anti-hIFNγ) conditions for 1 week and reactivated for further experiments, as previously described (10 (link)).
Webb L.M., Narvaez Miranda J., Amici S.A., Sengupta S., Nagy G, & Guerau-de-Arellano M. (2019). NF-κB/mTOR/MYC Axis Drives PRMT5 Protein Induction After T Cell Activation via Transcriptional and Non-transcriptional Mechanisms. Frontiers in Immunology, 10, 524.
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2

CD4+ T Cell Activation by HNSCC Exosomes

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Exosomes of HNSCC patients were co-incubated with healthy human CD4(+) T cells (105/100 µL) in the presence of 20 μM of exogenous ATP (Sigma Life Science). Exosome samples used were derived from the total, CD45(+), or CD45(−) fraction. The CD4(+) T cells were isolated using a CD4+ T cell isolation kit (17952, Stemcell Technologies) according to manufacturer’s instruction. Control samples incubated with no exosomes (PBS only) and without ATP were used.
After 20h of incubation, percentages of CD4+CD39+ T cells were determined by flow cytometry using anti-CD4-FITC Ab (11-0049-42, eBioscience) and anti-CD39-PECy7 Ab (25-0399-42, eBioscience, San Diego, CA, USA). The protocol was previously described in detail [8 (link),20 (link),31 (link)].
Beccard I.J., Hofmann L., Schroeder J.C., Ludwig S., Laban S., Brunner C., Lotfi R., Hoffmann T.K., Jackson E.K., Schuler P.J, & Theodoraki M.N. (2020). Immune Suppressive Effects of Plasma-Derived Exosome Populations in Head and Neck Cancer. Cancers, 12(7), 1997.
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3

Adoptive Transfer Model of Peanut Allergy

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We used an adoptive transfer model to examine the roles of PD-1 expressed on CD4+ T cells in the immune reaction to peanut allergens. Briefly, CD4+ T cells were isolated from splenocytes of naïve CD57BL/6 mice or pdcd1−/− mice by negative selection using a CD4+ T cell isolation kit (Stem Cell Technologies). Approximately 1×107 CD4+ T cells were transferred to naive Tcrb−/− mice by intravenous retroorbital injection. Starting 24 h after the T cell transfer, the mice were exposed to peanut flour through the airway for 4 weeks as described above.
Lama J.K., Iijima K., Kobayashi T, & Kita H. (2022). Blocking the inhibitory receptor PD-1 prevents allergic immune response and anaphylaxis in mice. The Journal of allergy and clinical immunology, 150(1), 178-191.e9.
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4

Primary T Cell Isolation and Analysis

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Primary mouse splenic T cells were isolated using a Pan T cell negative selection kit or a CD4+ T cell isolation kit (Stem Cell Technologies). Primary T cells or 2B4 cells were stained with fluorescently labeled Abs against GITR, CD4, CD8 (53–6.7), or FoxP3 (FJK-16S). For intracellular staining, cells were fixed and permeabilized with a fixation/permeabilization kit (BD Cytofix/Cytoperm, BD Biosciences). All flow cytometry and cell sorting were performed with Accuri C6 (BD Bioscience) in our own lab, or LSRII with UV (BD Bioscience), and Aria Fusion cell sorter (BD Bioscience) at the University of Iowa Flow Cytometry Facility, which is a Carver College of Medicine/Holden Comprehensive Cancer Center core research facility.
Li H., Hostager B.S., Arkee T, & Bishop G.A. (2021). Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR. The Journal of Biological Chemistry, 297(3), 101097.
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5

Dendritic Cell-Mediated T Cell Activation

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Bone marrow was obtained from the long bones of FcRγ−/− B6 mice and plated at a density of 106 cells per 6-well dishes in RPMI (Corning), 10 % FCS, 1 %l-Glutamine, streptomycin (100 mg/ml), and penicillin (100 U/ml). For the maturation of DCs, 40 ng/ml of both GM-CSF and IL-4 (PeproTech, Rocky Hill, NJ) was added to the media. Media were replaced after 3 days, and on the 6th day mature DCs were replated at a density of 5 × 104 per well in U-bottom 96 wells. Two μM of DTA-1 was administered to DCs for all in vitro experiments. For DC-pulsed T cell proliferation assays, BMDC was pulsed with OVA peptides for MHC-class I (SIINFEKL—1 μg/ml) or MHC-class II (ISQAVHAAHAEINEAGR—10 μg/ml) (Sigma) and 2 μM DTA-1. After 24 h, CD8 T cells from OTI mice (CD4+ from OTII mice) were obtained from splenocytes using a CD8 or CD4 T cell isolation kit, respectively (StemCell Technologies, Vancouver, CA), and stained with Cell Trace Violet (Life Technologies, Carlsbad, CA) as previously described [41 ]. After 72 h, cells were transferred to V-bottom 96 wells and stained for via flow cytometry.
Miska J., Rashidi A., Chang A.L., Muroski M.E., Han Y., Zhang L, & Lesniak M.S. (2016). Anti-GITR therapy promotes immunity against malignant glioma in a murine model. Cancer immunology, immunotherapy : CII, 65(12), 1555-1567.
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6

Isolation and Cultivation of Human Tregs

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Peripheral blood mononuclear cells (PBMCs) were isolated from donors by Ficoll-Paque PLUS (GE Healthcare) or Lymphoprep (Stemcell) gradient centrifugation. Total CD4+ T cells were isolated by negative magnetic selection using a CD4 T cell isolation kit (Stemcell) and CD4+CD25hiCD127lo-negCD45RO+ Treg cells were sorted on a FACS Aria (BD Biosciences). Treg cells were cultured in RPMI 1640 medium supplemented with 5% Human serum, 2 nM L-glutamine, 5 mM HEPES, and 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5 mM sodium pyruvate, 0.05 mM nonessential amino acids, and 5% human AB serum (Gemini Bio-Products). 96-well round bottom plates (Corning) were pre-coated with anti-human CD3 (UCHT1) (1–2 μg/ml) and used for Treg in vitro culture with soluble anti-human CD28 (28.2) (1–5 μg/ml) (BD Bioscience) and human IL-2 (50 U/ml). Human IL-2 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). TH1-Treg cells were induced with human recombinant IL-12 (20 ng/ml) (R&D). The Wnt/β-catenin inhibitor PKF115–584 (Tocris) was used at 200 nM and IWR-1 (Tocris) was used at 20 μM. SGK1 inhibitor GSK650394 (Tocris) was used at 10 μM. AKT inhibitor MK2206 (Tocris) was used at 5 μM. Frizzled 8 FC chimera protein (R&D) was used at 500 ng/ml.
Sumida T., Lincoln M.R., Ukeje C.M., Rodriguez D.M., Akazawa H., Noda T., Naito A.T., Komuro I., Dominguez-Villar M, & Hafler D.A. (2018). Activated β-Catenin in Foxp3+ Regulatory T cells Links Inflammatory Environments to Autoimmunity. Nature immunology, 19(12), 1391-1402.
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7

Passive Transfer of EAE in Mice

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CD4+ T cells were prepared from the spleen and inguinal lymph nodes of WT mice using a CD4+ T cell isolation kit (Stemcell Technologies; purity .97%). CD4+ T cells were injected i.v. (2 × 107 cells per mouse) into WT or RGC-32−/− mice. Five days later, the recipient mice were subjected to EAE induction. Rag1−/− mice were reconstituted with 7 × 106 CD4+ T cells from C57BL/6 or RGC-32−/− mice. One day later, EAE was induced in the recipient mice.
Rus V., Nguyen V., Tatomir A., Lees J.R., Mekala A.P., Boodhoo D., Tegla C.A., Luzina I.G., Antony P.A., Cudrici C.D., Badea T.C, & Rus H.G. (2017). RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis. Journal of immunology (Baltimore, Md. : 1950), 198(10), 3869-3877.
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8

Murine E.G7 T-cell Lymphoma Protocol

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The E.G7 T-cell lymphoma line was kindly provided by Dr. Jacques Galipeau (University of Wisconsin–Madison, WI, USA). All cell culture media and reagents were purchased from Wisent Bioproducts (St Jean-Baptiste, QC, Canada). The antibodies used in the flow cytometry, including I-Ab and CD47 antibodies, were purchased from BD Biosciences (San Jose, CA, USA). The IL-2 Quantikine kit was purchased from R&D Systems (Minneapolis, MN, USA). The chicken egg white ovalbumin (OVA) protein was purchased from Sigma-Aldrich (St-Louis, MI, USA). Recombinant murine IFNγ, GM-CSF and IL-21 were purchased from Peprotech (Rocky Hill, NJ, USA). The SIINFEKL and ISQAVHAAHAEINEAGR peptides were synthesized by Genscript (Piscataway, NJ, USA). The CD4 T-cell isolation kit was purchased from StemCell Technologies (Vancouver, BC, Canada). Liposomes and liposome-clodronate were purchased from Liposoma Research (Amsterdam, The Netherlands). XenoLight D-Luciferin K+ Salt was purchased from PerkinElmer (MA, USA). An Annexin-V staining kit was purchased from Cedarlane (Burlington, ON, Canada).
Bikorimana J.P., Abusarah J., Salame N., El-Hachem N., Shammaa R, & Rafei M. (2022). Humoral Immunity to Allogeneic Immunoproteasome-Expressing Mesenchymal Stromal Cells Requires Efferocytosis by Endogenous Phagocytes. Cells, 11(4), 596.
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9

Regulatory T Cell Transfer in Demyelination

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FoxP3-DTR mice were treated with DT as described above. In the 24 hr prior to lysolecithin-induced demyelination, mice were injected i.p. with 1x106 MACS-purified Treg from wild-type mice that were insensitive to DT treatment using a CD4+ T cell isolation kit (Stemcell Technologies).
Dombrowski Y., O’Hagan T., Dittmer M., Penalva R., Mayoral S.R., Bankhead P., Fleville S., Eleftheriadis G., Zhao C., Naughton M., Hassan R., Moffat J., Falconer J., Boyd A., Hamilton P., Allen I.V., Kissenpfennig A., Moynagh P.N., Evergren E., Perbal B., Williams A.C., Ingram R.J., Chan J.R., Franklin R.J, & Fitzgerald D.C. (2017). Regulatory T cells promote myelin regeneration in the Central Nervous System. Nature neuroscience, 20(5), 674-680.
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10

H-2K Specific T Cell Analysis

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Elicitation of H-2Kd specific Abs in BAL and serum following AM reconstitution were evaluated by staining mouse T cells (CD4+ T cell isolation kit, Stemcell Technologies) by BD FACSCanto II cell analyzer. Enumeration of H-2Kd and H-2Kb reactive T cells was conducted by stimulating splenocytes from recipient mice at 16 wk post AM-transfer with irradiated (30Gy) T cells from Balb/c and C57BL/6 respectively in ELISPOT assays (BD Biosciences).
Nayak D.K., Zhou F., Xu M., Huang J., Tsuji M., Hachem R, & Mohanakumar T. (2016). Long-term persistence of donor alveolar macrophages in human lung transplant recipients that influences donor specific immune responses. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(8), 2300-2311.
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