Cd45 magnetic beads

Manufactured by Miltenyi Biotec

Sourced in United States, Germany, Italy

CD45 magnetic beads are a laboratory product used for the isolation and enrichment of CD45-positive cells from biological samples. They consist of superparamagnetic particles coated with anti-CD45 antibodies. When a sample is exposed to these beads, the CD45-positive cells bind to the beads and can be separated magnetically.

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Lab products found in correlation

Automacs pro separator, by Miltenyi Biotec (3 mentions) Sterile yellow top tubes, by BD (2 mentions) Aria fusion, by BD (2 mentions) Automacs instrument, by Miltenyi Biotec (2 mentions) Rlt buffer, by Qiagen (2 mentions) α biotin magnetic beads, by Miltenyi Biotec (2 mentions) Liberase tm, by Roche (2 mentions) Lsrfortessa x 20, by BD (1 mentions) Octomacs, by Miltenyi Biotec (1 mentions) Astrios, by Beckman Coulter (1 mentions) Macs ls separation column, by Miltenyi Biotec (1 mentions) Anti cd3 antibody, by BioLegend (1 mentions) Cd31 clone 390, by Thermo Fisher Scientific (1 mentions) Vcam 1, by BD (1 mentions) Cd45 clone 30 f11, by BioLegend (1 mentions) Collagenase, by Fujifilm (1 mentions) Hanks balanced salt solution (hbss), by PAN Biotech (1 mentions) Ariaiii cell sorter, by BD (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) 70 μm cell strainer, by BD (1 mentions)

16 protocols using cd45 magnetic beads

Platelet Purification from Peripheral Blood

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Peripheral blood was collected in acid citrate-dextrose (ACD, 3.2%) sterile yellow-top tubes (Becton, Dickinson and Co.) and was processed within 4 h of collection for all samples. Platelets were isolated by established^{46 (link)–49} purification protocols. Briefly, the ACD-tube whole blood was first centrifuged at 200xg for 20min at room temperature (RT). The platelet-rich plasma (PRP) was removed and Prostaglandin E1 was added to the PRP to prevent exogenous platelet activation. The PRP was then centrifuged at 1000xg for 20min at RT. The platelet pellet was re-suspended in warmed (37 deg C) PIPES saline glucose (PSG). Leukocytes were depleted using CD45⁺ magnetic beads (Miltenyi Biotec). Isolated platelets were further resuspended in Trizol or LDS buffer for RNA (PCR) and protein (Western Blot) analyses.

Mosale Seetharam S., Liu Y., Wu J., Fechter L., Murugesan K., Maecker H., Gotlib J., Zehnder J., Paulmurugan R, & Krishnan A. (2023). Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens. bioRxiv.

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Isolation and Analysis of T Cells from HCC Tissues

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Fresh mouse liver tissues were finely chopped and dissociated into single-cell suspensions. After removal of red blood cells and liver cells, the leukocytes were further purified using a magnetic-activated cell-sorting separator with CD45 magnetic beads (Miltenyi Biotec, CA, USA). After incubation with V450-labeled CD3, PerCP-Cy™-labeled CD4, and V500-labeled CD8 (BD Biosciences, CA, USA), tumor-infiltrated T cells were detected by a flow cytometer (BD LSRFortessa X-20). Gating strategy for CD4⁺ and CD8⁺ T-cell in HCC tissues: lymphocytes were gated by forward and side scatter properties, and then CD4⁺/CD8⁺ T-cells were gated for further analysis (30 (link)).

Jiang Y., Chen S., Li Q., Liang J., Lin W., Li J., Liu Z., Wen M., Cao M, & Hong J. (2021). TANK-Binding Kinase 1 (TBK1) Serves as a Potential Target for Hepatocellular Carcinoma by Enhancing Tumor Immune Infiltration. Frontiers in Immunology, 12, 612139.

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Isolation and Purification of Mouse Cardiac Macrophages

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Mouse hearts were isolated and enzymatically digested, as previously described ^{5 (link)}, with the inclusion of 1mM Flavopiridol in the digestion buffer. Digestions were stopped after 20min and cells were processed into a single cell suspension on ice. Cells were stained with CD45 magnetic beads (Miltenyi Biotec) and subsequently fluorescently tagged antibodies against CD45, CD64 and CD11b. Hematopoietic cells were positively enriched using the AutoMacs instrument (Miltenyi Biotec) and live macrophages (CD45⁺, CD64⁺, CD11b⁺, DAPI^Neg) were sorted on the Aria Fusion (BD Bioscience) under low pressure into DMEM containing 50% FCS for cell visualization and 10X single cell RNA sequencing or RLT buffer (Qiagen) for RNA extraction.

Dick S.A., Macklin J.A., Nejat S., Clemente-Casares X., Momen A., Kantores C., Hosseinzadeh S., Barbu I., Chen J., Althagafi M.G., Besla R., Wong A., Aronoff L., Zaman R., Lavine K.J., Razani B., Ginhoux F., Husain M., Cybulsky M.I., Robbins C.S, & Epelman S. (2018). Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction. Nature immunology, 20(1), 29-39.

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Isolation of Lung Organoid Cells

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BAL fluid was collected from uninjured control or injured animals as described and red blood cells were lysed (0.154M NH₄Cl/10mM KHCO₃/0.127mM EDTA). The remaining cell suspension was incubated with CD45 magnetic beads (Miltenyi #130-052-301) and positively selected using the OctoMACS (Miltenyi) platform. Cells were then passed through a 0.4μm cell strainer prior to lung organoid co-culture.

Wong I.G., Stark J., Ya V., Moye A.L., Vazquez A.B., Dang S.M., Shehaj A., Rouhani M.J., Bronson R., Janes S.M., Rowbotham S.P., Paschini M., Franklin R.A, & Kim C.F. (2024). Airway injury induces alveolar epithelial and mesenchymal responses mediated by macrophages. bioRxiv.

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Enrichment of Hematopoietic and Stromal Cells from Tumor Tissue

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Hematopoietic cells from digested tumor suspensions were enriched by CD45 magnetic beads (Miltenyi Biotec) on an AutoMACS Pro Separator (Miltenyi Biotec) according to manufactures instructions. In some cases, hematopoietic depleted tissue suspensions were stained with α-gp38 (0.5 μg/mL) biotinylated antibody for 15 minutes at 4°C. Then, CAF and tissue resident fibroblasts were enriched from stained hematopoietic depleted suspensions by α-biotin magnetic beads (Miltenyi Biotec) on an AutoMACS Pro Separator (Miltenyi Biotec) according to manufactures instructions.

Rodriguez A.B., Peske J.D., Woods A.N., Leick K.M., Mauldin I.S., Meneveau M.O., Young S.J., Lindsay R.S., Melssen M.M., Cyranowski S., Parriott G., Conaway M.R., Fu Y.X., Slingluff CL J.r, & Engelhard V.H. (2021). Immune mechanisms orchestrate tertiary lymphoid structures in tumors via cancer-associated fibroblasts. Cell reports, 36(3), 109422.

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Isolation of Granulocytes and Myeloid Cells

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The whole blood from healthy donors and patients were processed as described above. We isolated the low-density granulocytes from the peripheral blood mononuclear layer and high-density granulocytes from the layer of white cells on the red cell pellet following Lymphoprep centrifugation by magnetic bead isolation using anti-CD15 microbeads (BD Pharmingen) and MACS LS separation columns (Miltenyi Biotech) according to the manufacturer’s instructions. Cell purity was >98% as confirmed by flow cytometry. Cell populations were similarly isolated from collagenase digested tumors using MACS beads (anti-CD15 for granulocytes and anti-CD14 for monocytes), followed by flow cytometric confirmation of purity. The dose of collagenase selected has previously been established to not affect cell surface marker expression or cell viability.
For isolation of autologous T cells and myeloid cells from the whole blood, the target populations were enriched first using positive selection with CD45 magnetic beads (Miltenyi Biotech) followed by staining with myeloid antibodies (above) and anti-CD3 antibody (BioLegend). Cells were sorted on Astrios (Beckman Coulter) using a 100 μm nozzle. DAPI was used as a viability marker to gate out the dead cells.

Khanna S., Graef S., Mussai F., Thomas A., Wali N., Yenidunya B.G., Yuan C., Morrow B., Zhang J., Korangy F., Greten T.F., Steinberg S.M., Stetler-Stevenson M., Middleton G., De Santo C, & Hassan R. (2018). Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(12), 2859-2872.

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Isolation and Characterization of Tumor Endothelial Cells

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Tumors were harvested 6 or 24 hours following FUS+MBs BTB/BBB opening. The tumors were minced and placed in digestion media containing 0.42U/ml Liberase TM (Roche). Samples were digested at 37 °C for 15 minutes and triturated every 5 minutes. Samples were homogenized (using glass homogenizers) and filtered through 70 µm filters. Subsequently, all samples were centrifuged at 1500 rpm for 15 minutes. The pellets were resuspended in CD45+ magnetic beads (Miltenyi Biotech) with Fc Block (1:1000) and incubated for 15 minutes at 4 °C. Samples were washed with AwesomeMacs Buffer and centrifuged at 1500 rpm for 5 minutes. Samples were separated with autoMACS Pro Separator with POSSEL AutoMACS protocol. The CD45 negative fraction was pelleted and stained with CD31 endothelial cell panel. Cells were Fc blocked and stained with fluorescent antibodies for CD31 (clone 390, eBioscience), CD45 (clone 30-F11, Biolegend), E-sel (clone 10E9.6, DB), P-selectin (clone RB40.34, DB), ICAM-1 (clone YNI/1.7.4, Biolegend), VCAM-1 (clone 429, BD) and Live/dead Aqua stain (eBioscience). Cells were fixed in 2% PFA.

Curley C.T., Stevens A.D., Mathew A.S., Stasiak K., Garrison W.J., Miller G.W., Sheybani N.D., Engelhard V.H., Bullock T.N, & Price R.J. (2020). Immunomodulation of intracranial melanoma in response to blood-tumor barrier opening with focused ultrasound. Theranostics, 10(19), 8821-8833.

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Platelet Isolation and Characterization

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Peripheral blood was collected in acid citrate-dextrose (3.2%) sterile yellow-top tubes (Becton, Dickinson and Co) and was processed within 4 hours of collection for all samples. Platelets were isolated by established45 (link), 46 (link), 47 (link), 48 (link) purification protocols. Briefly, the acid citrate-dextrose tube whole blood was first centrifuged at 200 g for 20 minutes at room temperature. The platelet-rich plasma (PRP) was removed, and prostaglandin E1 was added to the PRP to prevent exogenous platelet activation. The PRP was then centrifuged at 1000 g for 20 minutes at room temperature. The platelet pellet was resuspended in warmed (37°C) PIPES saline glucose. Leukocytes were depleted using CD45⁺ magnetic beads (Miltenyi Biotec). Isolated platelets were further resuspended in Trizol or LDS buffer for RNA (polymerase chain reaction [PCR]) and protein (western blot) analyses.

Seetharam S.M., Liu Y., Wu J., Fechter L., Murugesan K., Maecker H., Gotlib J., Zehnder J., Paulmurugan R, & Krishnan A. (2023). Enkurin: a novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens. Blood Advances, 7(18), 5433-5445.

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Isolation and Culture of Murine PαS MSCs

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Femura and tibiae were prepared as previously described with minor modifications [7 (link)]. The bone fragments were collected and digested for 1 h at 37 °C in alpha-MEM with L-Glutamine (PAN Biotech, Germany), 10 % FBS (PAA, Germany), 1 % penicillin/streptomycin (PAN Biotech) containing 3.92 U/ml collagenase (Wako Chemicals, Japan), 10 mM Hepes (Gibco, Germany) and 3 mM CaCl2. The cell suspension was filtered through a 70 μm cell strainer (BD Falcon, Germany) and collected by centrifugation at 400 g for 5 min at 4 °C. Red blood cells were lysed using 155 mM NH₄Cl/10 mM KHCO₃ buffer (pH 7.4) and washed with HBSS (PAN Biotech, Germany). After digestion, leucocytes were depleted with CD45 magnetic beads (Miltenyi Biotech, Germany) and stained with fluorochrome-labelled monoclonal antibodies and sorted by a BD ARIAIII cell sorter (Becton Dickinson, San Jose, CA, USA). PαS MSC were defined as positive for CD140a and Sca-1 and negative for CD45 and TER119 and were expanded in PureCoat Amine plates/flasks (BD, Germany) in alpha-MEM medium supplemented with L-Glutamine, 5 % FBS (mesenchymal stem cell-qualified, Life technologies, Germany), and 5 % human platelet lysate. Medium was changed every 3–7 days depending on cell growth. Human platelet lysate was prepared as previously described [24 (link)].

Hackstein H., Lippitsch A., Krug P., Schevtschenko I., Kranz S., Hecker M., Dietert K., Gruber A.D., Bein G., Brendel C, & Baal N. (2015). Prospectively defined murine mesenchymal stem cells inhibit Klebsiella pneumoniae-induced acute lung injury and improve pneumonia survival. Respiratory Research, 16, 123.

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Endothelial Cell Enrichment from Tumor

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Digested tumor suspensions were depleted of hematopoietic cells using CD45 magnetic beads (Miltenyi Biotec) on an AutoMACS Pro Separator (Miltenyi Biotec) according to manufacturer’s instructions. CD45⁺ depleted suspensions were stained with biotinylated anti-CD31 (0.5 µg/mL) for 15 minutes at 4°C and enriched using anti-biotin magnetic beads (Miltenyi Biotec) on an AutoMACS Pro Separator according to manufacturer’s instructions. BEC and TEC were sorted to highest purity according to the procedure below.

Rodriguez A.B., Parriott G, & Engelhard V.H. (2022). Tumor necrosis factor receptor regulation of peripheral node addressin biosynthetic components in tumor endothelial cells. Frontiers in Immunology, 13, 1009306.

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