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Mouse mdsc isolation kit

Manufactured by Miltenyi Biotec
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Sourced in Germany, United States

The Mouse MDSC Isolation Kit is a cell separation product designed to isolate mouse myeloid-derived suppressor cells (MDSCs) from various tissue samples. It utilizes magnetic bead-based separation technology to enrich the target MDSC population.

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Lab products found in correlation

Ls disposable column, by Miltenyi Biotec (2 mentions) Macs magnetic separator, by Miltenyi Biotec (2 mentions) Granulocyte macrophage colony stimulating factor, by Merck Group (2 mentions) Macrophage colony stimulating factor, by STEMCELL (2 mentions) Intesticult organoid growth medium, by STEMCELL (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Celecoxib, by LC Laboratories (2 mentions) Ah23848 hemicalcium salt, by LC Laboratories (2 mentions) Matrigel, by Corning (2 mentions) Dimethyl sulfoxide (dmso), by Corning (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) C57bl 6 mice, by Jackson ImmunoResearch (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions) Separation column, by Miltenyi Biotec (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Anti mouse cd11b beads, by Miltenyi Biotec (1 mentions) Collagenase 2, by Merck Group (1 mentions) Transwell insert, by Corning (1 mentions) Facsaria 2 sorp, by BD (1 mentions) T cell enrichment column, by R&D Systems (1 mentions)

23 protocols using mouse mdsc isolation kit

1

Isolation of Murine G-MDSCs and CD4+ T Cells

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Murine G-MDSCs were isolated from the bone marrow, spleen and tumor tissues of LCC tumor-bearing mice using a mouse MDSC isolation kit (Miltenyi Biotec, Auburn, CA). To improve the purity of G-MDSCs harvested from tumor tissues, Ly6G-enriched G-MDSCs were subsequently isolated using flow cytometry (FCM). In addition, murine CD4+ T cells were isolated from spleens of wild-type (WT) C57BL/6 mice using monoclonal anti-mouse CD4 antibodies conjugated to MicroBeads (Miltenyi Biotec, Auburn, CA). The purity of G-MDSCs and CD4+ T cells obtained from the isolated cells was confirmed by FCM.
Zheng Y., Tian X., Wang T., Xia X., Cao F., Tian J., Xu P., Ma J., Xu H, & Wang S. (2019). Long noncoding RNA Pvt1 regulates the immunosuppression activity of granulocytic myeloid-derived suppressor cells in tumor-bearing mice. Molecular Cancer, 18, 61.
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2

Splenic MDSC Depletion Protocol

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MDSCs were depleted from the splenocyte population of NBT1 tumor-bearing mice using a mouse MDSC Isolation Kit (Miltenyi Biotec, Auburn, CA) as per manufacturer’s instructions. Briefly, 1×108 splenocytes were resuspended in 350μl MACS buffer (PBS, 0.5% BSA, 2mM EDTA) and blocked for 10 minutes with 50μl FcR Blocking Reagent at 4°C. 100μl of anti-Ly6G-Biotin was added and incubated for 10 minutes at 4°C. Cells were washed in 10ml MACS buffer and resuspended in 100ul MACS buffer. 200μl anti-biotin microbeads were added and incubated at 4°C for 15 minutes. Magnetic separation was performed using LS disposable column and MACS Magnetic separator (Miltenyi Biotec). The negative fraction was collected and restimulated as described above. MDSC depletion was validated using flow cytometry.
de Vries C.R., Monken C.E, & Lattime E.C. (2015). The Addition of Recombinant Vaccinia HER2/neu to Oncolytic Vaccinia-GMCSF Given into the Tumor Microenvironment Overcomes MDSC-Mediated Immune Escape and Systemic Anergy. Cancer gene therapy, 22(3), 154-162.
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3

Isolation and Characterization of MDSCs from iKRAS Mice

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MDSCs were isolated from the spleens of iKRAS mice using a Mouse MDSC Isolation Kit (Miltenyi Biotec) and plated in RPMI1640 supplemented with 10% FBS and antibiotics. MDSCs (1×105 cells/well) were seeded in the top chamber of the transwell (Corning). Conditioned media from cultured iKRAS cells were collected and added to the bottom layer of the transwell. After 4h incubation, cells that had completely migrated to the bottom chamber were counted. A T cell suppression assay was performed as described previously [16 (link), 19 (link)] using equal numbers of MACS-sorted MDSCs and CFSE (Invitrogen)-labeled MACS-sorted (Miltenyi Biotec) CD8+ T cells in anti-CD3- and anti-CD28-coated 96 well plates at an MDSC/T cell ratio of 0:1, 1:1, and 4:1 with 5.0×105 MDSCs. MDSCs were isolated from iKRAS tumors and CD8+ T cells were isolated from the spleen of C57BL/6 mice (Jackson Labs). CFSE intensity was quantified 72 hours later with peaks identified by a BD LSRFortressa Cell Analyzer. CFSE peaks indicated the division times. Division times 0–2 and 3–4 were defined as low proliferation and high proliferation, respectively.
Gulhati P., Schalck A., Jiang S., Shang X., Wu C.J., Hou P., Ruiz S.H., Soto L.S., Parra E., Ying H., Han J., Dey P., Li J., Deng P., Sei E., Maeda D.Y., Zebala J.A., Spring D.J., Kim M., Wang H., Maitra A., Moore D., Clise-Dwyer K., Wang Y.A., Navin N.E, & DePinho R.A. (2022). Targeting T cell checkpoints 41BB and LAG3 and myeloid cell CXCR1/2 results in anti-tumor immunity and durable response in pancreatic cancer. Nature cancer, 4(1), 62-80.
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4

MDSC Subtype Isolation Protocol

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Isolation of CD11b+ granulocyte receptor 1–positive (Gr‐1+) total MDSCs, CD11b+ lymphocyte antigen 6 complex locus G‐negative (Ly6G‐) lymphocyte antigen 6 complex locus C‐high (Ly6Chi) Mo‐MDSCs, and CD11b+Ly6ClowLy6Ghi Gr‐MDSCs was performed by using mouse MDSC isolation kit (130‐094‐538; Miltenyi Biotech, Auburn, CA) according to the manufacturer’s instruction. Briefly, after Fc receptor blockade, cells were stained with biotin‐conjugated Gr‐1 or Ly6G antibody and further labeled with antibiotin microbeads. Labeled cells were passed through the separation columns (Miltenyi Biotech) for magnetic cell separation. Retained cells were analyzed for the CD11b+Gr‐1+/CD11b+Ly6G–Ly6Chi/CD11b+Ly6ClowLy6Ghi population to assess MDSC purity (>90%) by flow cytometry.
Li Y., Liu Z., Wang J., Yu J., Li Z., Yang H., Tang J, & Chen Z. (2019). Receptor‐Interacting Protein Kinase 3 Deficiency Recruits Myeloid‐Derived Suppressor Cells to Hepatocellular Carcinoma Through the Chemokine (C‐X‐C Motif) Ligand 1–Chemokine (C‐X‐C Motif) Receptor 2 Axis. Hepatology (Baltimore, Md.), 70(5), 1564-1581.
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5

Isolation and Culture of MDSCs

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Mice with B16 or H22 tumor were sacrificed and spleens were harvested, dissociated, and the red blood cells were lysed in lysis buffer. MDSCs were purified using a mouse MDSC isolation kit according to the manufacturer's instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purified cells were used for other experiments. MDSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) with 10% FBS enriched with 0.4 mmol/l of sodium pyruvate, 4 mmol/l of HEPES, and antibiotics (penicillin and streptomycin) in the Corning 3261 ultra-low attachment culture dishes (Corning Inc., Corning, NY, USA). Since MDSCs are incapable of survival without growth factor, granulocyte-macrophage CSF (GM-CSF) (40 pg/ml) was used to support cell viability and differentiation.
Gao Q., Jiang J., Chu Z., Lin H., Zhou X, & Liang X. (2017). Arsenic trioxide inhibits tumor-induced myeloid-derived suppressor cells and enhances T-cell activity. Oncology Letters, 13(4), 2141-2150.
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6

Isolation of Murine Myeloid-Derived Suppressor Cells

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The MDSCs isolated from the spleen and tumor tissue of tumor-bearing mice were isolated using a mouse MDSC isolation kit (Miltenyi Biotec, Auburn, CA, USA) following the manufacturer’s protocol. To isolate MDSCs from the spleen, the spleen of tumor-bearing mice was removed aseptically and then minced to obtain single-cell suspensions. The obtained cells were stained with anti-mouse-CD11b-beads (Miltenyi Biotec, Auburn, CA, USA) for 30 min and washed with PBS twice. Then, cells were subjected to isolation with a column. To isolate MDSCs from the tumor tissues of tumor-bearing mice, tumor tissues were cut into small pieces (1–2 mm3) and digested with collagenase II (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 2 h on a rotating platform to obtain single-cell suspensions. The obtained cells were stained with anti-mouse-CD11b-beads (Miltenyi Biotec, Auburn, CA, USA) for 30 min and washed with PBS twice. Then, cells were subjected to isolation with a column. The purity of MDSCs isolated from spleen and tumor tissues was greater than 90 and 80%, respectively.
Tian X., Ma J., Wang T., Tian J., Zhang Y., Mao L., Xu H, & Wang S. (2018). Long Non-Coding RNA HOXA Transcript Antisense RNA Myeloid-Specific 1–HOXA1 Axis Downregulates the Immunosuppressive Activity of Myeloid-Derived Suppressor Cells in Lung Cancer. Frontiers in Immunology, 9, 473.
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7

Purification and Co-culture of gMDSC and NK Cells

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gMDSC were purified using the Mouse MDSC isolation kit (Miltenyi). Purified gMDSC were seeded in triplicates in 96 V-bottom well plates at 1:1 ratio with purified NK cells. Following 24 h of co-culture in the presence of absence of a transwell insert (5.0 μm, Corning), NK cells were harvested and stained with anti-MSR1 (REA148), anti-CD36 (HM36) and anti-CD68 (FA-11) for flow cytometric analysis.
Niavarani S.R., Lawson C., Bakos O., Boudaud M., Batenchuk C., Rouleau S, & Tai L.H. (2019). Lipid accumulation impairs natural killer cell cytotoxicity and tumor control in the postoperative period. BMC Cancer, 19, 823.
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8

Isolation of Murine MDSCs

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CD11b+Gr-1+ MDSCs were isolated from the spleens of ESS mice using a FACSAria II SORP (Becton Dickinson) cell sorter (Miltenyi Biotec). M-MDSCs and PMN-MDSCs were isolated using a mouse MDSC isolation kit (Miltenyi Biotec) following the manufacturer’s protocol.
Tian J., Hong Y., Zhu Q., Zhou H., Zhang Y., Shen Z., Guo H., Zhang Y., Ai X., Zhao F., Rui K., Xu H, & Wang S. (2020). Mesenchymal Stem Cell Enhances the Function of MDSCs in Experimental Sjögren Syndrome. Frontiers in Immunology, 11, 604607.
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9

Splenic MDSC Isolation and Adoptive Transfer

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Splenic MDSCs were isolated using a mouse MDSC isolation kit (Miltenyi Biotec) following the manufacturer’s protocol. For adoptive transfer experiment, MDSCs isolated from 22- to 24-week-old C57BL/6 and MRL/lpr mice were injected into 8-week-old C57BL/6 mice (2 × 106 cells/mouse), once every 2 weeks for 10 weeks.
Yang Y., Zhang X., Jing L., Xiao Y., Gao Y., Hu Y., Jia S., Zhou G., Xiong H, & Dong G. (2024). MDSC-derived S100A8/9 contributes to lupus pathogenesis by promoting TLR7-mediated activation of macrophages and dendritic cells. Cellular and Molecular Life Sciences: CMLS, 81(1), 110.
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10

Murine T Cell-MDSC Coculture Assay

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Murine CD3+ T cells were isolated from the spleens of tumor-naïve C57BL6 or Balb/C mice using T Cell Enrichment Columns (R&D Systems, MTCC-25) and stained with carboxyfluorescein succinimidyl ester (CFSE, 1 μmol·L−1). Murine M-MDSCs were isolated from the bone marrow of B16F10 tumor-bearing C57BL6 and 4T1 tumor-bearing Balb/c mice or Alzet osmotic pump-implanted Balb/c mice using a Mouse MDSC Isolation Kit (Miltenyi Biotec 130-094-538). CD3+ T-cell purity after column enrichment was confirmed to be higher than 75% (Fig. S1c). T cells and MDSCs were cocultured in the presence of IL-2 (10 IU per mL) and Dynabeads® CD3/CD28 T-cell activators (bead:T-cell ratios = 0.5:1 or 0.2:1) for three days, followed by flow cytometric quantification of proliferating T cells.
Lee E.J., Lee K.J., Jung S., Park K.H, & Park S.I. (2023). Mobilization of monocytic myeloid-derived suppressor cells is regulated by PTH1R activation in bone marrow stromal cells. Bone Research, 11, 22.
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