Mojosort mouse cd8 t cell isolation kit

Manufactured by BioLegend

Sourced in United States, France

The MojoSort™ Mouse CD8 T Cell Isolation Kit is a laboratory tool designed to isolate CD8 T cells from mouse samples. The kit utilizes magnetic beads coated with antibodies specific to mouse CD8 T cells, allowing for their separation from other cell types in the sample.

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Lab products found in correlation

Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions) Mojosort mouse cd4 t cell isolation kit, by BioLegend (4 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions) Easysep mouse pe, by BioLegend (3 mentions) Interleukin 2 (il 2), by BioLegend (2 mentions) Red blood cell lysis buffer, by Thermo Fisher Scientific (2 mentions) Ifn γ, by Abcam (2 mentions) Anti cd3 cd28 conjugated dynabeads, by Thermo Fisher Scientific (2 mentions) Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (2 mentions) Biotin αcd44 im7, by BioLegend (2 mentions) Ionomycin, by BD (2 mentions) Gentamycin, by Quality Biological (2 mentions) Anti mouse cd3 clone 2c11, by BioXCell (2 mentions) Soluble anti mouse cd28 clone 37, by BioXCell (2 mentions) Fetal bovine serum (fbs), by Gemini Bio (2 mentions) β mercaptoethanol, by Merck Group (2 mentions) Golgistop, by BD (2 mentions) L glutamine, by Corning (2 mentions) Rpmi 1640, by Corning (2 mentions) Hepes, by Corning (2 mentions)

Close spelling variants of this product

CD8 T cell (2 citations)

58 protocols using mojosort mouse cd8 t cell isolation kit

Isolation and Functional Analysis of Mouse CD8+ T Cells

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C57BL/6 female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., People’s Republic of China. The animal handling and experimental procedures were carried out in compliance with the recommendations of the guide for the care and use of laboratory animals of the Ministry of Health, People’s Republic of China (62 (link)). The experimental protocol was approved by the Institutional Animal Care and Use Committee of Zhoukou Normal University on 10 July 2018 (authorization no. IACUC-henau-20180710). Molecular biology kits and enzymes were purchased from TaKaRa Biotechnology, Dalian, People’s Republic of China. Oxidized glutathione, reduced glutathione, l-arginine hydrochloride, and guanidine hydrochloride were purchased from Amresco BioLabs (Solon, OH). Mouse IFN-γ ELISpot Plus (HRP [horseradish peroxidase]) was purchased from MabTech (Cincinnati, OH), and the MojoSort mouse CD8⁺ T-cell isolation kit was purchased from BioLegend (San Diego, CA).

Wang Y., Gao M., Li X., Zhu W., Zhao M., Li J., Liu X., Cao L., Li S., Zhang S., Zhang L, & Fan S. (2023). Structural Analyses of a Dominant Cryptosporidium parvum Epitope Presented by H-2Kb Offer New Options To Combat Cryptosporidiosis. mBio, 14(1), e02666-22.

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Isolation and Activation of Splenic CD8+ T Cells

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Splenic CD8⁺ T cells were isolated by negative selection using the MojoSort Mouse CD8⁺ T Cell Isolation Kit (Biolegend) following the manufacturer's instructions. Isolated cells were plated at 2×10⁶ per mL in RPMI 1640 containing 2.0 mM L-glutamine and 25 mM HEPES, supplemented with 10 mM Sodium Pyruvate, nonessential amino acids, 100 U/ml penicillin/streptomycin, 55 μM β-ME, and 10% fetal calf serum (complete media). Cells were stimulated with 1 μg/mL ionomycin (Invivogen) and 1 ng/mL phorbol myristate acetate (PMA, Invivogen) for 7 days, with complete media supplemented with 200 U/mL recombinant human IL-2 added on days 3 and 6. Following stimulation, cells were resuspended at 5×10⁵ per mL in RPMI 1640 supplemented with 0.1% bovine serum albumin, and were incubated at 4°C for 60 min with or without 100 nM (±)-AMG 487 (R&D Systems). Cells were then plated in the top well of 96 well transwell plate (3 μm polycarbonate membrane pore, Corning). The bottom well of the plate contained RPMI 1640 supplemented with 0.1% BSA, either with or without recombinant mouse CXCL9 or CXCL10 (Biolegend). Cells were allowed to migrate for 1 hr at 37°C, prior to data collection with a MACSQuant VYB flow cytometer (Miltenyi Biotech) and analysis using FlowJo version 10.

de Mingo Pulido Á., Gardner A., Hiebler S., Soliman H., Rugo H.S., Krummel M.F., Coussens L.M, & Ruffell B. (2018). TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer. Cancer cell, 33(1), 60-74.e6.

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Mouse CD8+ T Cell Isolation and Culture

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For in vitro cultures, murine CD8 + T cells from male and female mice were isolated from single cell suspension of lymph nodes and spleen by negative selection using the MojoSort Mouse CD8 T cell isolation kit (BioLegend). Alternatively, CD8⁺ T cells were labeled with anti-CD8α-APC (Biolegend, clone 53–6.7) and positively enriched using anti-APC microbeads (Miltenyi Biotech). T cells were cultured in modified RPMI 1640 medium with physiological glucose concentration (100 mg/dL) by diluting standard RPMI 1640 medium (Gibco) with glucose-free RPMI medium (Roth). The medium was supplemented with 10% FBS (Sigma), 50 μM 2-mercaptoethanol (β-ME), 1% penicillin/streptomycin and 1% GlutaMAX-I (all Gibco), unless otherwise stated. Platinum-E retroviral packaging cell line (Cell Biolabs Inc.) was cultured in standard DMEM with 10% FBS (Sigma) and 1% penicillin and streptomycin (Gibco) at 37 °C with 5% CO₂. MC38^OVA and MC38^hCD19 colon carcinoma cells were grown in DMEM with 10% FBS (Sigma), 2 mM glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin, 2 mM Hepes and 0.1 mM non-essential amino acids (all Gibco) at 37 °C with 5% CO₂.

Wu H., Zhao X., Hochrein S.M., Eckstein M., Gubert G.F., Knöpper K., Mansilla A.M., Öner A., Doucet-Ladevèze R., Schmitz W., Ghesquière B., Theurich S., Dudek J., Gasteiger G., Zernecke A., Kobold S., Kastenmüller W, & Vaeth M. (2023). Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells through HIF-1α-mediated glycolytic reprogramming. Nature Communications, 14, 6858.

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Purifying and Transferring CD8+ T Cells

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CD8⁺ T cells were magnetically purified from the spleens of indicated donor mice by negative selection using MojoSort Mouse CD8 T cell Isolation kit (Biolegend) to >95% purity. Where indicated, CD8⁺ T cells were CTV-labeled (Invitrogen) before transfer. OT1 cells were counted on a Vi-CELL automated cell counter (Beckman Counter) and indicated OT1 cell numbers (5 × 10² or 5 × 10³) in 1 × PBS were transferred i.v. to recipient mice 1 day before or on the day of immunization or infection.

Ivanova D.L., Thompson S.B., Klarquist J., Harbell M.G., Kilgore A.M., Lasda E.L., Hesselberth J.R., Hunter C.A, & Kedl R.M. (2023). Vaccine adjuvant-elicited CD8+ T cell immunity is co-dependent on T-bet and FOXO1. Cell reports, 42(8), 112911.

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Evaluating Anti-Bv8 Antibody Effects on MDSC and T Cell Interactions

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Gr1+ cells and CD8+ T cells were isolated from the spleens of EMT6 tumor-bearing mice using positive isolation EasySep Mouse PE, BioLegend, and MojoSort™ Mouse CD8 T Cell Isolation Kit, respectively. The Gr1+ cells (2x10⁵ cells/ml) were seeded in 24 well plates and co-cultured with anti-Bv8 or IgG control antibodies (10 µg/ml) for 24 hours. Cells were then collected and immunostained for G-MDSCs or M-MDSCs. To analyze cell viability, the cells were stained with 7AAD and propidium iodide (PI) and analyzed by flow cytometry. In some experiments, Gr1+ cells (0.5x10⁶ cells/ml) and CD8+ T cells (0.5x10⁶ cells/ml) were co-cultured in a 24-well plate in the presence of anti-Bv8 or IgG control antibodies for 24 hours. Subsequently, the cells were collected and analyzed by flow cytometry to detect different immune cell states as indicated in the figure and in Table S1. The flow cytometry gating strategy is shown in Figure S8B. In some experiments, granzyme B was evaluated in the conditioned medium obtained from the co-cultured system analyzed by ELISA (R&D systems). All experiments were performed at least in three biological replicates.

Benguigui M., Vorontsova A., Timaner M., Levin S., Haj-Shomaly J., Deo A., Menachem R., Manobla B., Cooper T.J., Raviv Z, & Shaked Y. (2022). Bv8 Blockade Sensitizes Anti-PD1 Therapy Resistant Tumors. Frontiers in Immunology, 13, 903591.

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Neutrophil-mediated CD8+ T cell activation

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GR1⁺ and CD8⁺ T cells were isolated from the spleens of 4T1 tumor bearing mice using EasySep Mouse PE, and MojoSort™ Mouse CD8⁺ T Cell Isolation Kit (BioLegend, Cat# 480008), respectively. Ly6E^hi neutrophils were generated in-vitro as described above. The GR1⁺ cells and Ly6E^hi neutrophils (1x10⁶/ml) were cultured in serum-free medium for 24 hours to generate conditioned medium (CM). CD8⁺ T cells (0.5x10⁶/ml) were cultured with CM of GR1⁺ or Ly6E^hi neutrophils cells for 24 hours, after which the cells were washed and analyzed by flow cytometry for the evaluation of activated CD8⁺ T cells (CD8⁺/CD25⁺), effector CD8⁺ T cells (CD8⁺/CD44⁺/CD62L^-), Granzyme B⁺ and Ki67⁺ cells. In some experiments, CD8⁺ T cells (0.5x10⁶/ml) were cultured with conditioned medium of Ly6E^hi neutrophils together with neutralizing antibodies anti-IL12p40 (1μg/ml) or anti-IL23p19 (2μg/ml) (R&D systems, Cat# MAB4991 and Cat# AF1619, respectively). After 24 hours, the cells were collected, washed and analyzed by flow cytometry for the evaluation of activated CD8⁺ T cells (CD8⁺/CD25⁺). The experiments were performed using five biological repeats.

Benguigui M., Cooper T.J., Kalkar P., Schif-Zuck S., Halaban R., Bacchiocchi A., Kamer I., Deo A., Manobla B., Menachem R., Haj-Shomaly J., Vorontsova A., Raviv Z., Buxbaum C., Christopoulos P., Bar J., Lotem M., Sznol M., Ariel A., Shen-Orr S.S, & Shaked Y. (2024). Interferon-stimulated neutrophils as a predictor of immunotherapy response. Cancer Cell, 42(2), 253-265.e12.

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Isolation of CD8+ T-cells and B-cells from Murine Lung Tumors

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The Mojosort mouse CD8⁺ T-cell isolation kit (#480008,
Biolegend) was used to negatively isolate CD8⁺ T-cells per the
manufacture’s instruction from digested fresh lung tumor tissues of
CXCR2^myeΔ/Δ mice or littermate control
CXCR2^WT mice one week after 1×10⁶ PyMT cells
were intravenously injected. Lung tissues were digested with collagenase (320
U/mL), dispase (0.26 U/mL), and DNAase (5 mU/mL) at 37°C for one hour and
processed using the “m_impTumor_01.01” program on a Miltenyi
gentleMACS Dissociator. We obtained 87% purity of CD8⁺ T-cell with
this protocol. The isolated CD8⁺ T cells were cultured in RPMI 1640
medium (Catalog number 22400–089, Gibco) containing 0.5% FBS. In the same
way, B cells were also negatively isolated from the tumor tissues using the
Mojosort mouse pan B-cell isolation kit (#480051, Biolegend). We obtained 85%
purity of B-cells, and the B cells were cultured in Excellebrate B-cell media
(#CCM031, R&D Systems).

Yang J., Yan C., Vilgelm A.E., Chen S.C., Ayers G.D., Johnson C.A, & Richmond A. (2020). Targeted Deletion of CXCR2 in Myeloid Cells Alters the Tumor Immune Environment to Improve Antitumor Immunity. Cancer immunology research, 9(2), 200-213.

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CD8+ T Cell Cytotoxicity Assay

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Cell viability was determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, WI, USA). Briefly, after treatment, CD8⁺ T cells were isolated from all the treated mice splenocytes using the Mojo Sort mouse CD8 T cell Isolation Kit (480035, Biolegend) following the manufacturer’s protocol. The isolated CD8⁺ T cells (5 × 10⁴ cells in 50 μL of culture medium/well) were co-cultured with 4T1 cells at a ratio of 50:1 and incubated at 37°C in a humidified atmosphere of 5% CO₂. After 24 h, 20 μL of Cell Titer 96 Aqueous One Solution reagent was added to each well, and the plates were incubated at 37°C in 5% CO₂ for 2 h. After transferring 100 μL of incubation medium from each well into a new 96-well plate, the absorbance was measured at 490 nm.

Pan Y.C., Nishikawa T., Chang C.Y., Tai J.A, & Kaneda Y. (2020). CXCL2 combined with HVJ-E suppresses tumor growth and lung metastasis in breast cancer and enhances anti-PD-1 antibody therapy. Molecular Therapy Oncolytics, 20, 175-186.

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CD8+ T Cell Cytotoxicity Assay with MnCl2

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CD8⁺ T cells isolated from spleens of 6–8 weeks old OT-I mice were purified by MojoSort^™ Mouse CD8 T Cell Isolation Kit (Biolegend, 480008). CD8⁺ T cells were mixed with BMDC and incubated with pre-plated B16F10-OVA-GFP cells (CD8⁺ T:BMDC:Tumor = 2:1:2) with or without the indicated concentrations of MnCl₂ for 24 h. B6F10-OVA-GFP cells were analyzed by flow cytometry.

Lv M., Chen M., Zhang R., Zhang W., Wang C., Zhang Y., Wei X., Guan Y., Liu J., Feng K., Jing M., Wang X., Liu Y.C., Mei Q., Han W, & Jiang Z. (2020). Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy. Cell Research, 30(11), 966-979.

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Isolation and Activation of Naïve and Total CD8+ T Cells

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CD8⁺ T cells were prepared from spleen using a MojoSort Mouse CD8⁺ T cell isolation kit (cat#480035; BioLegend). In naïve CD8⁺ T (CD44^lowCD62L^high) cell preparation, biotin anti‐CD44 mAb (cat#103004; BioLegend) was added. In the case of total CD8⁺ T cells, anti‐CD44 mAb was not added. After isolation, the cells (7.5 × 10⁵) were stimulated with immobilized anti‐TCR‐β mAb (3 μg/mL, H57‐597; BioLegend) and anti‐CD28 mAb (1 μg/mL, 37.5; BioLegend) with IL‐2 (10 ng/mL; cat#575406; BioLegend) for 2 days. The cells were then transferred to a new plate and further cultured with IL‐2 (10 ng/mL) for 5 days.

Kakuda T., Suzuki J., Matsuoka Y., Kikugawa T., Saika T, & Yamashita M. (2023). Senescent CD8 + T cells acquire NK cell‐like innate functions to promote antitumor immunity. Cancer Science, 114(7), 2810-2820.

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