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Macs anti cd45 and anti ter119 microbeads

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MACS anti-CD45 and anti-Ter119 microbeads are lab equipment used for cell separation and isolation. The microbeads are coated with antibodies specific to the CD45 or Ter119 cell surface markers, allowing for the selection or depletion of cells expressing these markers.

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Lab products found in correlation

Dnase 1, by AppliChem (5 mentions) Hepes, by Lonza (3 mentions) Dispase 1, by Merck Group (2 mentions) Collagenase p, by Merck Group (2 mentions) Macs ls column, by Miltenyi Biotec (2 mentions) Gentlemacs based mechanical disruption, by Miltenyi Biotec (2 mentions) Hepes ph 7, by Lonza (2 mentions) Collagenase type p, by Merck Group (2 mentions) Collagenase type 2, by Merck Group (2 mentions)

5 protocols using macs anti cd45 and anti ter119 microbeads

1

Isolation of Murine Splenic Stromal Cells

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Spleens were harvested and perfused with RPMI 1640 medium containing 2% FCS, 20 mM Hepes (all from Lonza), 0.2 mg/ml Collagenase P (Sigma Aldrich), 0.8 U/ml Dispase I (Sigma Aldrich) and 100 µg/ml DNaseI (Applichem) with 22 G syringe. Samples were torn into smaller pieces and incubated at 37 °C for 30 min, with resuspension and collection of supernatant every 15 min to PBS containing 1% FCS and 10 mM EDTA (MACS buffer). To enrich fibroblastic stromal cells, hematopoietic and erythrocytes were depleted by incubating the cell suspension with MACS anti-CD45 and anti-TER119 microbeads (Miltenyi Biotec) and passing them through a MACS LS column (Miltenyi Biotec). Unbound single-cell suspensions were used for further flow cytometric analysis.
Cheng H.W., Onder L., Novkovic M., Soneson C., Lütge M., Pikor N., Scandella E., Robinson M.D., Miyazaki J.I., Tersteegen A., Sorg U., Pfeffer K., Rülicke T., Hehlgans T, & Ludewig B. (2019). Origin and differentiation trajectories of fibroblastic reticular cells in the splenic white pulp. Nature Communications, 10, 1739.
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2

Lung Lymphocyte and Stromal Cell Isolation

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Mice were sacrificed at the indicated time points and immediately perfused with PBS. Lung-infiltrating lymphocytes were isolated using mechanical disruption of the organ. For isolation of myeloid and stromal cells from the lung, the tissue was cut into small pieces, transferred into a 24-well dish filled with RPMI 1640 medium containing 2% FCS, 20 mM Hepes pH 7.2 (all from Lonza), 1 mg/ml Collagenase Type P (Sigma-Aldrich), 25 μg/ml DNaseI (AppliChem) and 1 mg/ml Collagenase Type II (Sigma-Aldrich) in combination with gentleMACS-based mechanical disruption (Miltenyi Biotech). After 30 minutes incubation at 37°C, cell suspensions were washed with PBS containing 0.5% FCS and 10 mmol/L EDTA. Stromal cell fraction enrichment was achieved by depleting hematopoietic and erythroid cells using MACS anti-CD45 and anti-Ter119 microbeads (Miltenyi Biotec).
Cupovic J., Ring S.S., Onder L., Colston J.M., Lütge M., Cheng H.W., De Martin A., Provine N.M., Flatz L., Oxenius A., Scandella E., Krebs P., Engeler D., Klenerman P, & Ludewig B. (2021). Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells. Nature immunology, 22(8), 1042-1051.
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3

Isolation of Splenic Stromal Cells

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Spleens were harvested and perfused with RPMI 1640 medium containing 2% FCS, 20 mM HEPES (all from Lonza), 0.2 mg per ml Collagenase P (Sigma Aldrich), 0.8 U per ml Dispase I (Sigma Aldrich) and 100 μg per ml DNaseI (Applichem) with 22G syringe. Samples were torn into smaller pieces and incubated at 37°C for 30 min, with resuspension and collection of supernatant every 15 min to PBS containing 1% FCS and 10 mM EDTA (MACS buffer). To enrich fibroblastic stromal cells, hematopoietic and erythrocytes were depleted by incubating the cell suspension with MACS anti-CD45 and anti-TER119 microbeads (Miltenyi Biotec) and passing them through a MACS LS column (Miltenyi Biotec). Unbound single cell suspensions were used for further flow cytometric analysis.
Chauveau A., Pirgova G., Cheng H.W., De Martin A., Zhou F.Y., Wideman S., Rittscher J., Ludewig B, & Arnon T.I. (2020). Visualization of T Cell Migration in the Spleen Reveals a Network of Perivascular Pathways that Guide Entry into T Zones. Immunity, 52(5), 794-807.e7.
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4

Lung Lymphocyte and Stromal Cell Isolation

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Mice were sacrificed at the indicated time points and immediately perfused with PBS. Lung-infiltrating lymphocytes were isolated using mechanical disruption of the organ. For isolation of myeloid and stromal cells from the lung, the tissue was cut into small pieces, transferred into a 24-well dish filled with RPMI 1640 medium containing 2% FCS, 20 mM Hepes pH 7.2 (all from Lonza), 1 mg/ml Collagenase Type P (Sigma-Aldrich), 25 μg/ml DNaseI (AppliChem) and 1 mg/ml Collagenase Type II (Sigma-Aldrich) in combination with gentleMACS-based mechanical disruption (Miltenyi Biotech). After 30 minutes incubation at 37°C, cell suspensions were washed with PBS containing 0.5% FCS and 10 mmol/L EDTA. Stromal cell fraction enrichment was achieved by depleting hematopoietic and erythroid cells using MACS anti-CD45 and anti-Ter119 microbeads (Miltenyi Biotec).
Cupovic J., Ring S.S., Onder L., Colston J.M., Lütge M., Cheng H.W., De Martin A., Provine N.M., Flatz L., Oxenius A., Scandella E., Krebs P., Engeler D., Klenerman P, & Ludewig B. (2021). Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells. Nature immunology, 22(8), 1042-1051.
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5

Isolation of Tumor-Associated Stromal Cells

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Mice were killed at the indicated time points, and tumor tissue was collected. Tumor-infiltrating hematopoietic cells were isolated by using mechanical disruption of the tissue. For isolation of stromal cells, the tissue was cut into small pieces and subjected to enzymatic digestion in RPMI 1640 medium containing 2% FCS, 20 mmol/L HEPES (all from Lonza), 1 mg/mL Collagenase Type P or II (Sigma-Aldrich), and 25 mg/mL DNaseI (AppliChem, Omaha, Neb) in combination with gentleMACS technology-based mechanical disruption (Miltenyi Biotech). After 30 minutes of incubation at 378C, cell suspensions were washed with PBS containing 0.5% FCS and 10 mmol/L EDTA. Hematopoietic and erythroid cells were depleted with MACS anti-CD45 and anti-Ter119 microbeads (Miltenyi Biotec) to enrich the stromal cell fraction.
Cheng H.W., Onder L., Cupovic J., Boesch M., Novkovic M., Pikor N., Tarantino I., Rodriguez R., Schneider T., Jochum W., Brutsche M, & Ludewig B. (2018). CCL19-producing fibroblastic stromal cells restrain lung carcinoma growth by promoting local antitumor T-cell responses. The Journal of allergy and clinical immunology, 142(4).
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