Mojosort mouse cd4 na ve t cell isolation kit

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The MojoSort™ Mouse CD4 Naïve T Cell Isolation Kit is a laboratory tool designed to isolate CD4+ naive T cells from mouse samples. It utilizes a magnetic-based separation method to purify the target cell population.

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Naïve T cell Mojosort (2 citations) MojoSort Mouse CD4 (2 citations)

22 protocols using mojosort mouse cd4 na ve t cell isolation kit

Allogeneic T Cell and BMDC Co-culture

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The allogeneic T cells were isolated from BALB/c mouse splenocytes using MojoSort™ Mouse Naïve CD4 T cell Isolation Kit (480,040, BioLegend, USA) and co-cultured with BMDCs at a ratio of 1:10 for 4 days. The levels of IFN-γ, IL-4, IL-5, and IL-10 were quantified on the culture supernatant using commercially available ELISA kits.

Guo F., Tang Y., Zhang W., Yuan H., Xiang J., Teng W., Lei A., Li R, & Dai G. (2022). DnaJ, a promising vaccine candidate against Ureaplasma urealyticum infection. Applied Microbiology and Biotechnology, 106(22), 7643-7659.

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Bone Marrow-Derived Macrophage and T Cell Protocols

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Bone marrow-derived macrophages (BMDMs) were generated from bone marrow cells harvested from male mice as previously described (26 (link)). Briefly, hemolyzed whole-bone marrow cells were cultured in RPMI 1640 medium (#R8758, Sigma-Aldrich, St. Louis, USA) containing 10% FCS (#51820, Biowest, Nuaille, France), 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μM 2-ME, 10 μM minimum nonessential amino acid solution, 100 mM sodium pyruvate, 10 mM HEPES and 20 ng/mL mM-CSF (#135-14391, Wako, Osaka, Japan) for 6 days. To stimulate BMDMs, 100 ng/mL lipopolysaccharide (LPS) (#L3024, Wako) was added to the culture supernatant. Naïve CD4⁺ T cells were isolated from the mouse spleen with the MojoSort Mouse Naïve CD4⁺ T Cell Isolation Kit (#480040, BioLegend, San Diego, USA) and were maintained in RPMI-based medium (the same medium as that used for BMDM generation but not containing M-CSF) in a plate coated with anti-CD3ϵ (clone; 145-2111C, BioLegend) and anti-CD28 (clone; 37.51, TONBO Bioscience) antibodies for 3 days. For the Treg-polarizing conditions, medium containing 3 ng/mL TGF-β1 (#763102, BioLegend) was used.

Nagata K., Nagase H., Okuzumi A, & Nishiyama C. (2021). Delta Opioid Receptor Agonists Ameliorate Colonic Inflammation by Modulating Immune Responses. Frontiers in Immunology, 12, 730706.

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In vitro Th Cell Differentiation and Interactions with hUC-MSCs

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Naïve mouse CD4 T cells were differentiated into Th1, Th2, and Th17 cells in vitro by using the CellXVivo Mouse Th1, Th2, and Th17 Cell differentiation kit (R&D Systems, USA) according to the manufacturer’s instructions, respectively. Briefly, naïve splenic CD4 T cells were isolated by using the MojoSort Mouse Naïve CD4 T Cell Isolation Kit (Biolegend) and then cultured in Th differentiation media for 6 days. hUC-MSCs were added on day 3 at a MSC/T cell ratio of 1:10 and the cell preparations were subjected to flow cytometric analysis 3 days later. Human PBMCs were cultured with hUC-MSCs at MSC/PBMC ratio of 1:10 for 24 h. The PBMCs were then subjected to quantitative polymerase chain reaction (qPCR) analysis.

Shin J.W., Ryu S., Ham J., Jung K., Lee S., Chung D.H., Kang H.R, & Kim H.Y. (2021). Mesenchymal Stem Cells Suppress Severe Asthma by Directly Regulating Th2 Cells and Type 2 Innate Lymphoid Cells. Molecules and Cells, 44(8), 580-590.

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In Vitro Induction of Murine Regulatory T Cells

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FoxP3^EGFP mice (Haribhai
et al., 2007) were acquired from Jackson (006772) and bred in our
facility at Stanford University. Splenocytes were harvested from
tumor-naïve female FoxP3^EGFP mice. Spleens were subjected
to mechanical dissociation on 70μm cell strainers and washed with
HBSS supplemented with 2% FBS (HBSSF). Erythrocytes were lysed with
Ammonium-Chloride-Potassium (ACK) lysing buffer. Magnetic isolation of
naïve CD4 T cells was performed using the MojoSort Mouse naïve
CD4 T Cell Isolation Kit (BioLegend, 480040) according to the
manufacturer’s instructions. Cell suspensions were stained for
lineage markers and sorted on a FACSAria II for the
GFP⁻lin⁻ cells. Sorting was
performed to ensure no preexisting Tregs (e.g., natural Tregs) remained
following magnetic isolation. 3×10⁵ lymphocytes were
combined with 1.5×10⁵ tumor cells (B16-F0, LN6-987AL, or
LN8-1198AR) and 3×10⁵ anti-CD3/CD28 Dynabeads (Thermo) in
100μL of RPMI-1640 supplemented with 10% FBS, 2mM L-glutamine, 15mM
HEPES, 14.3 mM 2-mercaptoethanol, 1mM Sodium Pyruvate, 1 × MEM
Non-Essential Amino Acids Solution (Thermo, 11140050), and 4ng/mL
hTGF-β1 (Peprotech). CD3/28 stimulation was performed to mimic TCR
ligation and antigen recognition. Following 72 hours of culture, cells were
washed and stained for analysis by flow cytometry as described above.

Reticker-Flynn N.E., Zhang W., Belk J.A., Basto P.A., Escalante N.K., Pilarowski G.O., Bejnood A., Martins M.M., Kenkel J.A., Linde I.L., Bagchi S., Yuan R., Chang S., Spitzer M.H., Carmi Y., Cheng J., Tolentino L.L., Choi O., Wu N., Kong C., Gentles A.J., Sunwoo J.B., Satpathy A.T., Plevritis S.K, & Engleman E.G. (2022). Lymph node colonization induces tumor-immune tolerance to promote distant metastasis. Cell, 185(11), 1924-1942.e23.

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Naïve CD4+ T cell Differentiation

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Naïve CD4⁺ T cells were purified using the MojoSort™ Mouse Naïve CD4⁺ T cell isolation kit (Cat. 480033, Biolegend) following the manufacturer’s protocol. Naïve CD4⁺ T cells were then activated and differentiated by plating at a density of 1x10⁶ cells per well on coated anti-CD3 (2µg/mL, Cat.BE0001-1, BioXcell) and anti-CD28 (2µg/mL, Cat. BE0015-1, BioXcell) 48-well plates for four days in 37 °C incubators with 5% CO₂ with IL-2. IL-12 and anti-IL-4 were added to growth media to promote Th1 differentiation. IL-4, anti-IFN-γ, and anti-IL-12 were added to growth media to promote Th2 differentiation. TGFβ, IL-6, IL-1β, IL-23, anti-IL-4, anti-IL-12, and anti-IFN-γ were added to growth media to promote Th17 differentiation. TGFβ, anti-IL-4, anti-IL-12, and anti-IFN-γ were added to growth media to promote iTreg differentiation. After differentiation, cells were collected, washed with PBS, and analyzed by flow cytometry.

Hu Y., Han L., Xu W., Li T., Zhao Q., Lu W., Sun J, & Wang Y. (2024). CARD11 regulates the thymic Treg development in an NF-κB-independent manner. Frontiers in Immunology, 15, 1364957.

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Differentiation of Naive CD4+ T Cells

Cited 1 time

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Naive CD4⁺ T cells were obtained from spleen and peripheral lymph nodes of CRL and IF1-KO mice by negative selection using the MojoSort™ Mouse CD4 Naïve T Cell Isolation Kit (BioLegend). For in vitro activation, naive CD4⁺ T cells were incubated in the presence of 2 μg/ml plastic-bound purified anti-CD3, 1 μg/mL soluble anti-CD28, and 5 ng/mL recombinant mouse IL-2 during 48 h. For Th1 differentiation, culture medium was also supplemented with IL-12 (10 ng/mL) and anti-IL-4 (4 μg/mL). For Treg cell differentiation, TGF-β (10 ng/mL) was also added to culture medium. For Th17 differentiation, IL-2 was replaced and anti-IL-4 (5 μg/mL), anti-IFN-γ (5 μg/mL), TGF-β (5 ng/mL), IL-23 (20 ng/mL), and IL-6 (20 ng/mL) were incorporated in the culture medium.⁴³ (link) Cells were differentiated for 4 days in a humidified incubator at 37°C with a controlled atmosphere of ambient air 10% CO₂.

Romero-Carramiñana I., Dominguez-Zorita S., Esparza-Moltó P.B, & Cuezva J.M. (2024). Ablation of Atp5if1 impairs metabolic reprogramming and proliferation of T lymphocytes and compromises mouse survival. iScience, 27(6), 109863.

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Tracking Naïve CD4 T Cell Activation

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Naïve Thy1.1⁺ LCMV GP₆₁₋₈₀-specific (SMARTA, LCMV GP-specific) CD4 T cells were negatively sorted with MojoSort™ Mouse CD4 Naïve T cell Isolation Kit (BioLegend) according to the manufacturer's protocol. To sort CFSE^hi/lo cells, a FACSAria cell sorter (BD Biosciences) was used. One million sorted naïve CD4 T cells were transferred intravenously. At the indicated time points mice were immunized intraperitoneally with 100 μg LCMV-GP₆₁₋₈₀ peptide (LCMV-gp61, synthesized by Genecust) and 10 μg LPS (O111:B4, Sigma) in 200 μl PBS. To test cell division, naïve Thy1.1⁺ LCMV GP-specific CD4 T cells were labeled with 5 μM CFSE according to the manufacturer's protocol (BioLegend) followed by adoptive transfer and immunization.

Sarkander J., Hojyo S., Mursell M., Yamasaki Y., Wu T.Y., Tumes D.J., Miyauchi K., Tran C.L., Zhu J., Löhning M., Hutloff A., Mashreghi M.F., Kubo M., Radbruch A, & Tokoyoda K. (2020). Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location. Frontiers in Immunology, 10, 3113.

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Eosinophil-Mediated T Cell Modulation

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Flow-cytometry-purified BM-Eos (B6J) or magnetically enriched splenic eosinophils (Il5-tg) or A-Eos and B-Eos sorted from the GI tract (Il5-tg) were isolated from 6–12-week-old female and male mice. BM-Eos or spleen-derived eosinophils were conditioned overnight with colon CM (1:10) or treated with recombinant mouse IFNγ (10 ng ml⁻¹, PeproTech) and/or IL-33 (20 ng ml⁻¹, PeproTech), as indicated. Naive CD4⁺ T cells were isolated from the lymph nodes of 6–12-week-old female and male mice (B6J), enriched with the MojoSort Mouse CD4 Naïve T Cell Isolation Kit (480040 BioLegend) and purified by flow cytometry. T cells were labelled with the CellTrace CFSE Cell Proliferation Kit (C34554 Thermo Fisher Scientific) following the manufacturer’s instructions. T cells were then activated by CD3/CD28 T-activator Dynabeads (11131D Gibco) and co-cultured with eosinophils at a 1:1 ratio (2 × 10⁵ total) for 4 days at 37 °C in complete RPMI medium supplemented with 10 ng ml⁻¹ recombinant mouse IL-5 (PeproTech) and 20 ng ml⁻¹ IL-2 (402-ML R&D). CFSE dilution was assessed by flow cytometry.

Gurtner A., Borrelli C., Gonzalez-Perez I., Bach K., Acar I.E., Núñez N.G., Crepaz D., Handler K., Vu V.P., Lafzi A., Stirm K., Raju D., Gschwend J., Basler K., Schneider C., Slack E., Valenta T., Becher B., Krebs P., Moor A.E, & Arnold I.C. (2022). Active eosinophils regulate host defence and immune responses in colitis. Nature, 615(7950), 151-157.

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Naïve T Cell Activation and Treg Differentiation

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Mice were sacrificed, their spleens were removed and gently dissociated into single-cell suspensions. Naïve T cells were isolated using the MojoSort Mouse CD4 Naïve T Cell Isolation Kit (BioLegend). T cells were cultured in U-bottom 96-well plates with RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 2 mM l-glutamine, 1% antibiotic-antimycotic (Gibco), 10 mM HEPES (Gibco), 1× nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), and 55 μM β-mercaptoethanol (Gibco). Naïve T cells were activated with plate-bound anti-CD3 (3 μg/ml; Bio X Cell) and soluble anti-CD28 (2 μg/ml; Bio X Cell) antibodies. T_reg cell differentiation was achieved by the indicated concentration of recombinant human TGF-β (R&D). The Akt inhibitor MK-2206 and afuresertib were purchased from MedChemExpress (MCE). Cells were cultured for 72 h and detected by flow cytometry.

Cai W., Zhang J., Zhou H., Li X., Lou F., Sun Y., Xu Z., Bai J., Yin Q., Wang Z., Sun L., Cai X., Tang S., Wu Y., Fan L., Wang H., Wang H, & Li Q. (2021). Protein phosphatase 6 (Pp6) is crucial for regulatory T cell function and stability in autoimmunity. Genes & Diseases, 9(2), 562-575.

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Splenic Dendritic Cell Isolation and T Cell Enrichment

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Spleens were harvested from mice and mechanically dissociated using frosted microscope slides then rinsed with 1X PBS. Single cell suspensions were passed through 70-µm cell strainers and red blood cells were then lysed with ammonium chloride-potassium (ACK) lysis buffer. Cells were counted with hemacytometer and 3–5 × 10⁶ cells were used per antibody-staining reaction. For experiments requiring enrichment of splenic cDCs, cells were enriched from total spleen cells with MojoSort™ Mouse CD11c Nanobeads (BioLegend Cat: 480078) according to manufacturer’s protocol. After positive selection cells were counted via hemacytometer to assess viability and cell count with purity assessed via flow cytometry. For adoptive transfer and co-culture experiments naïve OT-II cells were magnetically enriched with MojoSort™ Mouse CD4 Naïve T Cell Isolation Kit (BioLegend Cat: 480040) according to manufacturer’s protocol. After positive selection cells were counted via hemacytometer to assess viability and cell count with purity assessed via flow cytometry.

Flynn P.A., Long M.D., Kosaka Y., Long N., Mulkey J.S., Coy J.L., Agarwal A, & Lind E.F. (2024). Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency. Frontiers in Immunology, 15, 1297338.

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