Clone 37

Manufactured by BioXCell

Clone 37.51 is a versatile laboratory equipment designed for cloning and gene expression applications. It features a compact and efficient design to facilitate precise and reliable experimental procedures. The core function of Clone 37.51 is to enable the isolation, amplification, and manipulation of genetic material, supporting a wide range of research and development activities.

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Lab products found in correlation

Clone 145 2c11, by BioXCell (5 mentions) Recombinant mouse il 12, by Thermo Fisher Scientific (2 mentions) Na ve cd4 t cell isolation kit, by STEMCELL (2 mentions) Recombinant human mouse rat activin a, by R&D Systems (2 mentions) Recombinant mouse il 6, by Thermo Fisher Scientific (2 mentions) Easysep mouse na ve cd8 t cell isolation kit, by STEMCELL (1 mentions) Facsaria 2, by BD (1 mentions) Anti il 4, by BioXCell (1 mentions) Anti cd28, by BioXCell (1 mentions) Recombinant il 12, by Thermo Fisher Scientific (1 mentions) Retronectin, by Takara Bio (1 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (1 mentions) Murine il 2, by BioLegend (1 mentions) Easysep mouse t cell isolation kit, by STEMCELL (1 mentions) Calphos mammalian transfection kit, by Takara Bio (1 mentions) Polybrene, by Merck Group (1 mentions)

7 protocols using clone 37

Isolating and Activating Murine CD8+ T cells

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For in vivo transfer, CD8⁺ T-cells were first purified from the spleen using the EasySep™ Mouse CD8α Positive Selection Kit II (STEMCELL Technologies Inc, Vancouver, BC), then naïve OT-I cells were further sorted as CD8⁺Vβ5.1⁺CD44⁻CD62L⁺ using FACS Aria II (BD Bioscience, San Jose, CA). For memory CD8⁺ T-cell transfer, memory OT-I cells were sorted as CD8⁺CD45.2⁺Vα2⁺CD44⁺CD62L⁺ on an Aria II, routinely to higher than 98% purity. For in vitro activation and stimulation, naïve CD8⁺ T-cells were enriched from the spleen and peripheral lymph nodes using the EasySep™ Mouse Naïve CD8⁺ T Cell Isolation Kit (STEMCELL Technologies Inc, Vancouver, BC), routinely to 90– 95% purity.
For polyclonal activation, naïve CD8⁺ T-cells were stimulated with plate-bound anti-CD3 (1 μg/ml) (Clone 145-2C11, BioX Cell, West Lebanon, NH) and soluble anti-CD28 (1 μg/ml) (Clone 37.51, BioX Cell, West Lebanon, NH) Abs for the indicated times. For antigen-specific T-cell activation, splenocytes (1 × 10⁶/ml) from OT-I CK2α^fl/fl or OT-I CK2α^−/− mice were cultured in R10 medium, incubated with OVA_257–264 peptide (0.1 ng/ml) (InvivoGene, San Diego, CA), and then cells were harvested at the indicated time points.

Yang W., Wei H., Benavides G.A., Turbitt W.J., Buckley J.A., Ouyang X., Zhou L., Zhang J., Harrington L.E., Darley-Usmar V.M., Qin H, & Benveniste E.N. (2022). Protein Kinase 2 (CK2) Controls CD8+ T-cell Effector and Memory Function during Infection. Journal of immunology (Baltimore, Md. : 1950).

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Th1 Cell Differentiation and Memory

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Naive T cells (CD4⁺ CD44⁻) were polarized into Th1 cells for 3 days using plate-bound anti-CD3 (5 µg/ml, clone 145-2C11, BioXCell), anti-CD28 (1 µg/ml, clone 37.51, BioXCell), recombinant IL-12 (10 ng/ml, Peprotech) and anti-IL-4 (10 µg/ml, BioXCell). Th1 cells were then cultured, for 4 to 7 additional days, into IL-7-supplemented (20 ng/ml, Peprotech) media to obtain memory-like cells.

Lepez A., Pirnay T., Denanglaire S., Perez-Morga D., Vermeersch M., Leo O, & Andris F. (2020). Long-term T cell fitness and proliferation is driven by AMPK-dependent regulation of reactive oxygen species. Scientific Reports, 10, 21673.

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Differentiation of CD4+ T Helper Cell Subsets

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For CD4⁺ T helper (T_H) cell differentiation, purified naive CD4⁺CD25⁻CD62L^hiCD44^lo T cells from wild-type C57BL/6 (B6) mice or CD4⁺CD25⁻CD62L^hiCD44^loThy1.1⁻ T cells from Foxp3^Thy1.1 mice (1 × 10⁶/ml) were activated with plate-bound αCD3 (1 μg/ml; 1452C11; Bio Xcell) and αCD28 (2 μg/ml; clone 37.51; Bio Xcell) antibodies in the presence of appropriate cytokines. T_H0: anti-IL-4 (10 μg/ml), anti-IFN-γ (10 μg/ml; XMG 1.2; Bio Xcell) and 100U/ml of recombinant human IL-2(rhIL-2), T_H17: rIL-1β (20 ng/ml), rIL-6 (20 ng/ml), hTGF-β1 (2 ng/ml), anti-IL-4 (10 μg/ml) and anti-IFN-γ (10 μg/ml) or iT_reg: rhIL-2(100U/ml) and human TGF-β1 (hTGF-β1; 5 ng/ml) conditions in T cell media. 100 U/ml of rhIL-2 (T_H0 and iT_reg) and 30 U/ml of rhIL-2 and rIL-23 (10 ng/ml) (T_H17) were added on 3 days after detaching from αCD3/αCD28 antibodies, and the cells were expanded in complete T cell media additional for 2 days.

Hwang S.M., Sharma G., Verma R., Byun S., Rudra D, & Im S.H. (2018). Inflammation-induced Id2 promotes plasticity in regulatory T cells. Nature Communications, 9, 4736.

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Naïve CD4+ T Cell Polarization

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Naïve CD4⁺ T cells were isolated from spleen using the Naïve CD4⁺ T cell isolation kit (Stem Cell). Purity was 92% or higher. Naïve CD4⁺ T cells (2 × 10⁵ cells/well) were cultured for 3 d with recombinant human/mouse/rat activin A (50 ng/ml, R&D Systems), recombinant mouse IL-12 (10 ng/ml, Peprotech), and/or recombinant mouse IL-6 (20 ng/ml, Peprotech) in the presence of plate-bound anti-mouse-CD3 mAb (8 µg/ml, clone 145-2C11, BioXcell) and anti-mouse-CD28 (8 µg/ml, clone 37.51, BioXcell), in RPMI medium, supplemented with 10% fetal bovine serum, GlutaMAX, penicillin/streptomycin, and 2.5 µM β-mercaptoethanol. After 3 d, cells were removed from stimuli and further cultured for 2 additional d in IL-2 (50 U/ml,) and the same cytokine combination used at day 0. Phenotype was quantified by flow cytometry (Supplementary Table 3) at day 5 of the in vitro culture.

Locci M., Wu J., Arumemi F., Mikulski Z., Dahlberg C., Miller A.T, & Crotty S. (2016). Activin A programs human TFH cell differentiation. Nature immunology, 17(8), 976-984.

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Suppressive Potency of GITR+CD25+ CD4+ Cells

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For evaluating suppressive potency, CD4 splenocytes were initially enriched by negative selection (Miltenyi) from KLF2^GFP reporter or KLF2^GFP FOXP3^RFP dual reporter mice, stained with anti-CD4, anti-CD8 and anti-CD44 antibodies, followed with sorting (BD FACS-Aria). For evaluating suppression by GITR+CD25⁺ cells, mLN CD4 cells were enriched from CD4^Cre KLF2^f/f or Cre-negative littermate control mice, stained anti-CD4, anti-CD8, anti-GITR, and anti-CD25 antibodies, and sort purified as GITR+CD25⁺ CD4⁺ (CD8-negative) cells. Thereafter, suppression was assayed by co-culture with 10⁵ CFSE-labeled (5μM for 10 min at room temperature) responder splenocytes from congenially discordant (CD45.1 or CD90.1) mice on the B6 background, or BG-2 TCR transgenic mice, and stimulation with anti-CD3 (2.5 μg/mL, clone 145–2C11, BioXcell) and anti-CD28 (1.25 μg/mL, clone 37.51, BioXcell) or β-galactosidase peptide (500nM) or with each indicated neutralizing or isotype antibody (50 μg/mL) in 200 μL DMEM medium (supplemented with 10% fetal bovine serum, 1% L-glutamine, 10 mM HEPES, 1% penicillin-streptomycin) at 37°C with 5% CO₂ for 96 h.

Shao T.Y., Jiang T.T., Stevens J., Russi A.E., Troutman T.D., Bernieh A., Pham G., Erickson J.J., Eshleman E.M., Alenghat T., Jameson S.C., Hogquist K.A., Weaver C.T., Haslam D.B., Deshmukh H, & Way S.S. (2023). Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation. Cell reports, 42(11), 113323.

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Murine T Cell Transduction Protocol

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To produce ecotropic retrovirus for murine T cell transduction, Phoenix-ECO cells were transfected with a 1:3 ratio of pCL-Eco and transfer plasmid using a CalPhos Mammalian Transfection Kit (Takara Bio). Primary murine T cells were isolated from spleens of WT or CD45.1⁺ C57BL/6 mice using the EasySep Mouse CD8⁺ T Cell Isolation Kit or EasySep Mouse T Cell Isolation Kit (StemCell Technologies) and activated for 48 h on six-well non-tissue culture (TC)-treated plates precoated with 0.5 mg ml⁻¹ anti-CD3 (BioXCell, Clone 2C11) and 5 mg ml⁻¹ anti-CD28 (BioXCell, Clone 37.51) at 10⁶ cells ml⁻¹ with 5 ml per well. T cells were cultured in complete RPMI with 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol and 1× minimal essential medium non-essential amino acids (ThermoFisher) supplemented with 10 ng ml⁻¹ murine IL-2 (BioLegend). Following activation, T cells were transduced by spinfection at 3 × 10⁶ cells per well in complete T cell medium for 90 min at 1,100g and 32 °C with 10 ng ml⁻¹ polybrene (Sigma) on non-TC-treated plates pre-coated with 15 mg ml⁻¹ RetroNectin (Takara Bio). CAR expression was assayed via flow cytometry 24 h post-transduction; T cells were maintained at a cell density of 10⁶ cells ml⁻¹, then used for adoptive transfer or in vitro assays at 48 h post-transduction.

Zhang A.Q., Hostetler A., Chen L.E., Mukkamala V., Abraham W., Padilla L.T., Wolff A.N., Maiorino L., Backlund C.M., Aung A., Melo M., Li N., Wu S, & Irvine D.J. (2023). Universal redirection of CAR T cells against solid tumours via membrane-inserted ligands for the CAR. Nature Biomedical Engineering, 7(9), 1113-1128.

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Naïve CD4+ T Cell Polarization

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Locci M., Wu J., Arumemi F., Mikulski Z., Dahlberg C., Miller A.T, & Crotty S. (2016). Activin A programs human TFH cell differentiation. Nature immunology, 17(8), 976-984.

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