Dynabeads untouched mouse cd4 cell kit

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The Dynabeads® Untouched™ Mouse CD4 cell kit is a magnetic bead-based system used for the isolation of untouched mouse CD4+ T cells from a single-cell suspension. The kit utilizes a negative selection approach to separate the CD4+ T cells from other cell types without affecting their phenotype or functionality.

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Dynabeads (2112 citations) Dynabeads Untouched Mouse CD4 Cells Kit (48 citations) CD4 Dynabeads (14 citations) Dynabeads Untouched Mouse CD4+ T cells Kit (4 citations) Dynabeads Untouched Mouse CD4 Cells negative selection kit (2 citations)

9 protocols using dynabeads untouched mouse cd4 cell kit

Baicalin Modulates Regulatory T Cell Differentiation

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Naive CD4⁺T cells were isolated from spleens of MRL/lpr mice by using the Dynabeads Untouched Mouse CD4 Cell Kit (Life Technologies AS, Norway). For Tfh cell differentiation, naive CD4⁺T cells were cultured for 5 days with 10 ng/ml IL-21 (PeproTech, USA), 10 ng/ml IL-6 (PeproTech), and 25 µl of anti-CD3/anti-CD28 antibody (BD Biosciences Pharmingen, USA) with various doses of Baicalin (dissolved in DMSO and diluted with PBS) or a DMSO-PBS vehicle as a control. For Foxp3⁺ regulatory T cell differentiation, sorted naive CD4⁺T cells were cultured for 5 days with 5 ng/ml TGF-β (PeproTech), 30 U/ml IL-2 (PeproTech, USA), and 50 ng/ml anti-CD3/anti-CD28 antibody (BD Biosciences Pharmingen) with or without Baicalin as described above. For some differentiation experiments, 10 μM mammalian target of rapamycin (mTOR) agonist (MHY1485; MedChem Express, USA),or 200 ng/ml mTOR antagonist rapamycin (LC Laboratories) was added with 40 μM Baicalin for 5 days.
For some experiment, sorted naive CD4⁺T cells were cultured for 5 days with 5 ng/ml TGF-β, 30 U/ml IL-2, and 50 ng/ml anti-CD3/anti-CD28 antibody with or without 40 μM Baicalin for 5 days, then these 1 × 10⁶ Baicalin-induced Foxp3⁺ regulatory T cells or 1 × 10⁶ vehicle-induced Foxp3⁺ regulatory T cells were injected intravenously into MRL/lpr mice weekly for 4 weeks.

Yang J., Yang X., Yang J, & Li M. (2019). Baicalin ameliorates lupus autoimmunity by inhibiting differentiation of Tfh cells and inducing expansion of Tfr cells. Cell Death & Disease, 10(2), 140.

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Adoptive Transfer of Splenic CD4+CD45RBhigh T Cells

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Using a well-established protocol [3 (link)], splenic CD4⁺CD45RB^high T cells from C57BL/6 WT mice were adoptively transferred into same-sex 8–12-week-old Rag1^tm1Mom mice. First, the spleens from C57BL/6 WT mice were removed and placed in FACS buffer (PBS, fetal bovine serum, and sodium azide). Then, the spleens were homogenized by rubbing between glass slides until a single-cell suspension was created. The single-cell suspension was passed through a 70µm cell strainer. Cells were enriched by negative isolation using Dynabeads Untouched Mouse CD4 Cell Kit (Life Technologies AS, Carksbad, CA, USA) according to the manufacturer’s instructions. CD4 cells were fluorescently labeled with fluorescein isothiocyanate (FITC) rat anti-mouse CD45RB (Thermo Fischer Scientific, Waltham, MA, USA) and phycoerythrin (PE) rat anti-mouse CD4 (BD Biosciences, Franklin Lakes, NJ, USA). CD4+ T cells were sorted using a Becton-Dickenson Influx cell sorter by double gating on CD4⁺ singlet cells and the brightest 40% CD45RB^high cells. Cells were sorted into tubes containing post-sorting buffer (PBS, EDTA, FBS) to stabilize the post-sorted T cells. In total, 5 × 10⁵ CD4⁺CD45RB^high T cells were then injected intraperitoneally into recipient Rag1^tm1Mom mice of the same sex as the T cell donors.

Maltz R.M., Marte-Ortiz P., McClinchie M.G., Hilt M.E, & Bailey M.T. (2024). T Cell-Induced Colitis Is Exacerbated by Prolonged Stress: A Comparison in Male and Female Mice. Biomedicines, 12(1), 214.

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Retroviral Infection of Activated CD4+ T Cells

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CD4⁺ T cells were purified from the spleen and lymph nodes using the Dynabeads® Untouched™ Mouse CD4 Cell Kit (Life Technologies). Purified CD4⁺ T cells were stimulated overnight using plate-bound anti-CD3ε and anti-CD28 antibodies (5µg/ml each). In the case of LLO118 T cells, 10µM LLO_190-205 peptide was applied to mixed lymphocytes from mouse lymph node to stimulated CD4⁺ T cells. Spin infection of stimulated T cells using retrovirus was performed at 1258g for 90min at 37°C. Virus-infected cells were then used for in vitro or in vivo experiments.
The PPIF shRNA-silencing vectors were generated by ligating PPIF shRNA into the MSCV plasmid. The shRNA sequence for PPIF sh665 and PPIF sh710 (shPPIF) can be found in Supplemental Experimental Procedures.

Zhang B., Liu S.Q., Li C., Lykken E., Jiang S., Wong E., Gong Z., Tao Z., Zhu B., Wan Y, & Li Q.J. (2016). MicroRNA-23a curbs necrosis during early T cell activation by enforcing intracellular reactive oxygen species equilibrium. Immunity, 44(3), 568-581.

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Antigen-Specific T Cell Proliferation

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CD4⁺ T cells from BALB/c DO11.10 mice were negatively selected using Dynabeads untouched mouse CD4 cell kit (Life Technologies, Grand Island, NY, USA). Cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; eBioscience) and 10⁷ cells in 200 μl PBS were injected intravenously into naïve BALB/c mice. After 2 days, virosomes, liposomes, or PBS was administrated intranasally as described above. Three days later, LDLNs and NDLNs were collected and stained for surface markers and intracellular cytokines as mentioned above. Antigen-specific T cell proliferation (CFSE dilution) and cytokine production were measured by flow cytometry and analyzed with FlowJo9 software (TreeStar).

Blom R.A., Amacker M., van Dijk R.M., Moser C., Stumbles P.A., Blank F, & von Garnier C. (2017). Pulmonary Delivery of Virosome-Bound Antigen Enhances Antigen-Specific CD4+ T Cell Proliferation Compared to Liposome-Bound or Soluble Antigen. Frontiers in Immunology, 8, 359.

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Isolation and Activation of Murine CD4+ T Cells

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CD4⁺T cells were isolated from the total lymphocytes population in the lymph nodes (LNs) using a Dynabeads^® Untouched™ Mouse CD4 cell kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, cells were incubated with an antibody mixture for 20 min. Bead-bound cells were then incubated for 15 min in Dynabeads buffer. The bead-free cells were then transferred to a new tube and resuspended in fresh medium. The isolated CD4⁺ T cells were cultured in RPMI 1640 (Gibco, Uxbridge, UK) containing L-Glutamine and 25 mM HEPES and supplemented with 10% (vol/vol) FBS (Gibco), 100 U/mL penicillin-streptomycin (Gibco), and 0.05 mM 2-β-mercapto-ethanol. For the cytokine analysis, isolated CD4⁺T cells (1.0 × 10⁶ cells/mL) were incubated in the presence or absence of PMA (10 ng/mL) and Ionomycin (1 μg/mL) with indicated concentrations of APO-9 (6.25 to 50 μM) for 4 hr.

Han S.C., Kang N.J., Yoon W.J., Kim S., Na M.C., Koh Y.S., Hyun J.W., Lee N.H., Ko M.H., Kang H.K, & Yoo E.S. (2016). External Application of Apo-9′-fucoxanthinone, Isolated from Sargassum muticum, Suppresses Inflammatory Responses in a Mouse Model of Atopic Dermatitis. Toxicological Research, 32(2), 109-114.

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CD4+ T-cell Purification from Lung and Lymph Nodes

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For leukocytes prepared from lung tissue, CD4⁺ T-cells were purified by FACS, using a BioRad S3e Cell Sorter. For cells from the mesenteric and mediastinal lymph nodes, CD4⁺ T-cells were purified using Dynabeads Untouched Mouse CD4 Cell kit (Invitrogen).

Lorentsen K.J., Cho J.J., Luo X., Zuniga A.N., Urban JF J.r., Zhou L., Gharaibeh R., Jobin C., Kladde M.P, & Avram D. (2018). Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma. Nature Communications, 9, 1679.

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Adoptive Transfer of NR1 T Cells

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For naive NR1 transfers, CD4⁺ T cells were isolated from secondary lymphoid organs from RFP WT NR1 mice, RFP IFN-γ^−/− NR1 mice, or RFP IFN-γR^−/− NR1 mice. For Th1-skewed NR1 T cells, CD4⁺ T cells were purified from secondary lymphoid organs of RFP WT NR1 mice, RFP IFN-γ^−/− NR1 mice, or RFP IFN-γR^−/− NR1 mice using a Dynabeads Untouched mouse CD4 cell kit (Invitrogen). T cells were stimulated using irradiated splenocytes from a C57BL/6 mouse that had been pulsed with 5 μM Cta1 peptide (Cta1_133-152) at a 4:1 stimulator/T cell ratio. Cells were cultured for 5 days in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum, l-glutamine, HEPES, 50 mM 2-mercaptoethanol, 50 U/ml penicillin, and 50 mg/ml streptomycin along with 10 ng/ml interleukin-12 (IL-12; Peprotech, Rocky Hill, NJ) and 10 μg/ml anti-IL-4 (Bio X Cell, West Lebanon, NH). A total of 10⁶ total naive or Th1-skewed NR1 T cells were injected intravenously in a 200-μl volume into recipient mice 1 day prior to infection with C. trachomatis.

Helble J.D., Gonzalez R.J., von Andrian U.H, & Starnbach M.N. (2020). Gamma Interferon Is Required for Chlamydia Clearance but Is Dispensable for T Cell Homing to the Genital Tract. mBio, 11(2), e00191-20.

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Murine CD4+ T Cell Differentiation

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Purified CD4⁺ T cells were isolated from C57BL/6 splenocytes using the Dynabeads untouched mouse CD4 Cell kit (Invitrogen) according to the manufacturer’s instructions. Purified CD4⁺ T lymphocytes were cultured in RPMI Medium 1640 with GlutaMAX supplemented with 10 % FBS, 1 units/ml penicillin/0.1 mg/ml streptomycin, 1 mM sodium pyruvate, 20 mM HEPES, and 50 μM of β-mercaptoethanol (Invitrogen, Grand Island, NY, USA). T cells were stimulated in 48-well plates coated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) antibodies (BD Biosciences, San Diego, CA, USA) and were subjected to specific T-cell differentiation. Th1 differentiation was induced by adding 5 ng/ml of IL-12 and 2.5 μg/ml of anti-IL-4. Th17 differentiation was induced by adding TGF-β (5 ng/ml), IL-6 (50 ng/ml), and IL-23 (5 ng/ml) and neutralizing antibodies for IFN-γ and IL-4 (2.5 μg/ml each). Each stimulation period lasted 5 days. T cells were plated into the wells, and MSCs were added in a 1:10 ratio (MSCs:CD4⁺ T cells). For FACS analysis, differentiated Th1 and Th17 cells were restimulated with PMA/ionomycin for 3.5 hours in the presence of brefeldin for the last 2.5 hours of incubation at 37 °C before antibody staining.

Vega-Letter A.M., Kurte M., Fernández-O’Ryan C., Gauthier-Abeliuk M., Fuenzalida P., Moya-Uribe I., Altamirano C., Figueroa F., Irarrázabal C, & Carrión F. (2016). Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE. Stem Cell Research & Therapy, 7, 150.

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Analyzing STAT5 Phosphorylation in iTregs

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To assess STAT5 phosphorylation, iTreg cells differentiated in the presence of TGFβ alone, TGFβ+ VitC or TGFβ+ RA + VitC were collected and washed on day 6 after differentiation. 1.5–2 × 10⁶ cells were plated in 100 μl culture medium in a round‐bottom 96‐well plate. The cells were rested for 1 h at 37°C and then mixed with 100 μl of 2X IL‐2 cytokine mix to get a final concentration of 0, 3, 5, 10, and 100 U/ml of IL‐2. The cells were then restimulated with IL‐2 at 37°C for 1 h. After restimulation, cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences) at room temperature for 10 min. The fixed cells were washed twice with MACS buffer, permeabilized by adding 100 μl of ice‐cold True‐Phos Perm Buffer (BioLegend), and incubated for at least 1 h at −20°C. The cells were then washed twice with MACS buffer, stained with mouse anti‐Stat5 (pY694) (Clone: 47/Stat5 (pY694); Alexa Fluor 647; 10 µl/assay; catalog #612599; BD Biosciences) and analyzed by flow cytometry on LSR‐II. To examine STAT5 phosphorylation in endogenous Treg cells, CD4⁺ T cells were isolated from Tet2^fl/flTet3^fl/fl Foxp3‐eGFP CD4CRE mice and littermate controls using Dynabeads Untouched Mouse CD4 Cell Kit (Invitrogen) and used for IL‐2 restimulation (10 and 100 U/ml of IL‐2). CD4⁺Foxp3⁺ Treg cells were gated to examine the level of STAT5 phosphorylation.

Yue X., Samaniego‐Castruita D., González‐Avalos E., Li X., Barwick B.G, & Rao A. (2021). Whole‐genome analysis of TET dioxygenase function in regulatory T cells. EMBO Reports, 22(8), e52716.

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