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Mojosort

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MojoSort is a magnetic cell separation system that can be used to isolate specific cell populations from complex samples. It operates on the principle of magnetic separation, allowing for the efficient and gentle isolation of target cells.

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Lab products found in correlation

Easysep, by STEMCELL (3 mentions) β actin clone 13e5, by Cell Signaling Technology (2 mentions) Pten 138g6, by Cell Signaling Technology (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Anti cd3ε antibody, by BD (1 mentions) Etomoxir, by Merck Group (1 mentions) Rotenone antimycin a, by Agilent Technologies (1 mentions) Mitotracker, by Thermo Fisher Scientific (1 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Rhil 2, by BioLegend (1 mentions) Anti cd3, by BioLegend (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Ficoll, by GE Healthcare (1 mentions) Anti cd28, by BioLegend (1 mentions) Granulocyte macrophage colony stimulating factor, by Immunotools (1 mentions) Rhil 12 p70, by Thermo Fisher Scientific (1 mentions) Fastpure total rna isolation kit, by Vazyme (1 mentions)

28 protocols using mojosort

1

Naïve T Cell Enrichment

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Primary T cells were enriched by magnetic cell sorting to more than 95% purity by using MojoSort (Biolegend). To enrich naïve T cells, an optimal dose of magnetic beads bound to anti-CD44 mAb (Biolegend, 10 μg mAb/100 μL beads) was mixed with T cells and incubated for 15 min. Then, the samples were subjected to magnetic sorting to deplete CD44hi effector cells.
Ueda Y., Higasa K., Kamioka Y., Kondo N., Horitani S., Ikeda Y., Bergmeier W., Fukui Y, & Kinashi T. (2023). Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1. iScience, 26(8), 107292.
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2

Bone Marrow Mast Cell Desensitization

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About 95% purity of bone marrow MCs (BMMCs) were obtained as previously described11 (link) (Supplementary Fig. 5). Cells were sensitized overnight with anti-dinitrophenyl (DNP) IgE (0.25 μg/mL). The next day, the cells were washed to eliminate the excess of unbound IgE and resuspended at 37 °C in 100 μL of fresh medium with 5 ng/mL IL-3. For desensitization, cells were treated as described in the previous report.38 (link) Briefly, DNP-human serum albumin (HSA) was added every 10 min for desensitization in 200 μL culture condition (50 pg, 250 pg, 250 pg, 500 pg, 500 pg, 1 ng, 2 ng, 8 ng, 16 ng, 17.5 ng); the cells were then stimulated with 80 ng for 1 h.38 (link) For in vitro coculture analysis, MCs were pre-sensitized with anti-DNP IgE (0.25 μg/mL) overnight without antigen; they were then co-cultured for 3 days at 37 °C in PBS with 8 × 105 CD4 + T cells isolated from the spleen and mesenteric lymph nodes with MojoSort (Biolegend); the plates were pre-coated with 0.125 µg/mL anti-CD3ε antibody (BD Pharmingen).
Takasato Y., Kurashima Y., Kiuchi M., Hirahara K., Murasaki S., Arai F., Izawa K., Kaitani A., Shimada K., Saito Y., Toyoshima S., Nakamura M., Fujisawa K., Okayama Y., Kunisawa J., Kubo M., Takemura N., Uematsu S., Akira S., Kitaura J., Takahashi T., Nakayama T, & Kiyono H. (2020). Orally desensitized mast cells form a regulatory network with Treg cells for the control of food allergy. Mucosal Immunology, 14(3), 640-651.
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3

Optimizing CD4+ T Cell Activation and Transduction for CAR-T Therapy

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CD4+ T cells were isolated from processed C57BL/6 spleens by magnetic bead selection and typically had a purity of >90% (Miltenyi, San Diego, CA or MojoSort, Biolegend, San Diego, CA). The CD4+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 antibodies at a concentration of 500 ng/mL each for 48 h at 37 °C. T cells were transduced with virus to express the B7H6-specific CAR and mouse T-bet, T-bet (STOP), or T-bet (∆TBOX) constructs through spin transduction, as previously described [40 (link)]. Mock T cells were generated to serve as a negative control. Mock T cells are CD4+ T cells which have been transduced with a pFB-neo viral vector which contains no gene construct insertion. Concanavlin A (ConA)-activated CAR T cells were transduced with B7H6-specific CAR retrovirus, as previously described [40 (link)]. CAR T cells underwent G418 selection for 3 days at 0.5 mg/mL then histopaque enriched to isolate live cells. Purified CD4+ T cells and ConA-stimulated T cells were cultured in complete RPMI 1640 media with 100 U/mL or 25 U/mL of rIL-2, respectively.
Gacerez A.T, & Sentman C.L. (2018). T-bet promotes potent antitumor activity of CD4+ CAR T cells. Cancer Gene Therapy, 25(5), 117-128.
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4

Metabolic Profiling of Antigen-Specific CD8 T Cells

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For Seahorse analysis, antigen-specific CD8 T cells were purified from splenocytes using EasySep (Stemcell Technologies) or MojoSort (BioLegend) positive biotin selection kits. Cells were plated at 4x105 (XF 24) or 1.5x105 (XF 96) per well using poly-L-lysine adhesive (Sigma). Agilent XF 24 and 96 analyzers were utilized. Oligomycin, Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), Rotenone/Antimycin A (Agilent Seahorse XF Cell Mito Stress Test Kit), 2-deoxyglucose (VWR), and etomoxir (Sigma-Aldrich) were used to probe different metabolic assays. Mitochondrial assays were performed with samples after isolation using tetramethylrhodamine ethyl ester (TMRE) and Mitotracker (Invitrogen) per manufacturer’s instructions. Fluorescent glucose analog N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) (Invitrogen) was used for glucose flux analyses, and 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene (BODIPY 493/503) (Invitrogen) was used for fatty acid content analyses per manufacturer’s instructions. For metabolomics analysis, cells were FACS purified and stored in 80% methanol to quench metabolism. Cytosolic metabolites were isolated and dried using Speedvac. Samples were sent to the Northwest Metabolomics Research Center for LC/TQ MS. Data were analyzed at Elucidata.
Kalia V., Yuzefpolskiy Y., Vegaraju A., Xiao H., Baumann F., Jatav S., Church C., Prlic M., Jha A., Nghiem P., Riddell S, & Sarkar S. (2021). Metabolic regulation by PD-1 signaling promotes long-lived quiescent CD8 T cell memory in mice. Science translational medicine, 13(615), eaba6006.
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5

Isolation and Culture of Primary B Cells

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Human peripheral blood mononuclear cells (PBMCs) were enriched from blood of healthy donors by Ficoll density gradient centrifugation. CD19+ cells (B cells) were freshly isolated from PBMCs using either the MojoSort (Biolegend) or EasySep (Stem Cell Tech) B cell isolation kit by means of negative selection with magnetic cell sorting (MACS), plated at 5 × 104–106 cells/well and allowed to rest for 1 h prior to stimulation experiments. Primary cells were cultivated in IMDM supplemented with 10% FCS (Thermo Fisher). Ramos and BJAB B cells were cultivated in RPMI 1640 supplemented with 10% FCS, 1% L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher).
Frensch M., Jäger C., Müller P.F., Tadić A., Wilhelm I., Wehrum S., Diedrich B., Fischer B., Meléndez A.V., Dengjel J., Eibel H, & Römer W. (2021). Bacterial lectin BambL acts as a B cell superantigen. Cellular and Molecular Life Sciences, 78(24), 8165-8186.
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6

Activation of CD8+ T Cells by TAMs

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Ficoll (GE Healthcare) was applied to separate human PBMCs. CD8+ T cells were isolated from purified PBMCs or tumour tissue‐digested single‐cell suspensions by human CD8 nanobeads (MojoSort, BioLegend). During a 4‐day incubation, bead‐purified peripheral CD8+ T cells (1 × 105 cells/well in 24‐well plates) were co‐cultured with IgG2B‐treated TAMs or α‐VISTA‐treated TAMs isolated from tumour tissues in 1 mL Roswell Park Memorial Institute (RPMI) 1640 (Gibco) with 10% FBS (Gibco), rhIL‐2 (80 ng/mL, BioLegend), anti‐CD3 (2 μg/mL, BioLegend) and anti‐CD28 (1 μg/mL, BioLegend) antibodies (Figure 6G‐H). After 4‐day incubation, CD8+ T cells were harvested for flow cytometry (FC) or intracellular flow cytometry (ICFC).
Cao Y., Yu K., Zhang Z., Gu Y., Gu Y., Li W., Zhang W., Shen Z., Xu J, & Qin J. (2024). Blockade of V‐domain immunoglobulin suppressor of T‐cell activation reprograms tumour‐associated macrophages and improves efficacy of PD‐1 inhibitor in gastric cancer. Clinical and Translational Medicine, 14(2), e1578.
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7

Cytokine Profiling of Stimulated Monocyte-Derived Dendritic Cells

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Preparation of monocyte‐derived dendritic cells was performed by harvesting MNCs as described above and subsequent isolation of CD14+ monocytes by magnetic sorting (MojoSort, BioLegend). MoDCs were generated by addition of interleukin 4 and granulocyte‐macrophage colony stimulating factor (both 500 U/mL, ImmunoTools, Germany) to monocytes in 6‐well plates for 7 days as described previously, harvested, and resuspended in complete RPMI for stimulation experiments. MoDCs were grown and incubated in a humidified atmosphere of 5 % carbon dioxide at 37 °C. For the stimulation, compounds were added to moDCs (1x106/mL) in the indicated concentrations and incubated for 20 h. Cytokine releases of stimulated moDCs were quantified after 20 h by using commercial ELISA Kits (Life Technologies GmbH, Darmstadt, Germany) specific for IL‐6, IL‐10 and IL‐12‐p70 in supernatants. Data were combined from n=3 independent experiments; error bars indicate standard error of the mean.
Strobl S., Hofbauer K., Heine H, & Zamyatina A. (2022). Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants. Chemistry (Weinheim an Der Bergstrasse, Germany), 28(35), e202200547.
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8

Stimulation of Naive CD4 T Cells

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Naive CD4 T cells of healthy controls were purified using MojoSort (BioLegend), stimulated at a concentration of 5 µl/106 cells/ml anti-CD3/CD28/CD2 beads (STEMCELL Technologies SARL) in X-VIVO 15 medium (Lonza Sales Ltd.) + 5% antibody (AB) serum (Sigma-Aldrich) and 100 ng/ml rhIL-12p70 (PeproTech) until further use.
Kögl T., Chang H.F., Staniek J., Chiang S.C., Thoulass G., Lao J., Weißert K., Dettmer-Monaco V., Geiger K., Manna P.T., Beziat V., Momenilandi M., Tu S.M., Keppler S.J., Pattu V., Wolf P., Kupferschmid L., Tholen S., Covill L.E., Ebert K., Straub T., Groß M., Gather R., Engel H., Salzer U., Schell C., Maier S., Lehmberg K., Cornu T.I., Pircher H., Shahrooei M., Parvaneh N., Elling R., Rizzi M., Bryceson Y.T., Ehl S., Aichele P, & Ammann S. (2024). Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect. The Journal of Experimental Medicine, 221(7), e20221122.
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9

Isolation of Highly Pure Mouse Neutrophils

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Mouse neutrophils were purified from bone marrow cells using negative magnetic bead selection (MojoSort, BioLegend). In brief, bone marrow cells were flushed from femurs and tibias using sterile RPMI 1640 medium supplemented with 10% FBS and 2 mM EDTA onto a 50 mL screw top Falcon tube fitted with a 100 μm filter (Swamydas et al., 2015 (link)). Cells were washed with 1X MojoSort buffer (1X PBS, 0.5% BSA, 2mM EDTA). Neutrophils were isolated according to the manufacturer’s instructions. Bone marrow-enriched neutrophils had > 90% purity and > 90% viability as determined by flow cytometry (in vitro experiments). In the adoptive transfer experiments, the purity exceeded 94%.The remaining ~10% of cells were identified as CD45+ CD19+ (B cells) < 2.72%, CD45+ CD3+ (T cells) < 0.24%, CD45+ Ly6Chi (inflammatory monocytes) < 0.31%, CD45+ MHC II+ (dendritic cells) < 2.37%, and CD45− (stromal cells) < 4.00%.
Swidergall M., Solis N.V., Wang Z., Phan Q.T., Marshall M.E., Lionakis M.S., Pearlman E, & Filler S.G. (2019). EphA2 Is a Neutrophil Receptor for Candida albicans that Stimulates Antifungal Activity during Oropharyngeal Infection. Cell reports, 28(2), 423-433.e5.
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10

CD4+ Cell Signaling Pathway Analysis

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For cell signaling molecules, total CD4+ cells were isolated from secondary lymphoid tissues of mice using MojoSort™ magnetic separation (BioLegend). Cells were activated with anti-CD3/28 ± RA (20 nM) or left unactivated for indicated times. Proteins were extracted from cells with a lysis buffer (1% Triton X-100 and 0.1% SDS in PBS) and probed using antibodies for total Akt (9272), pAkt (T308; 244F9), pS6K (T421/S424; CST #9204), PTEN (138G6), and β-actin (clone 13E5), which were purchased from Cell Signaling Technology.
Friesen L.R., Gu B, & Kim C.H. (2020). A ligand-independent fast function of RARα promotes exit from metabolic quiescence upon T cell activation and controls T cell differentiation. Mucosal immunology, 14(1), 100-112.
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