Ls003086

Manufactured by Worthington

LS003086 is a laboratory equipment product. It is a device designed for use in scientific and research environments. The core function of this product is to perform specific tasks or measurements as required for laboratory experiments and analysis.

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Lab products found in correlation

Ls003126, by Worthington (1 mentions) P4937, by GE Healthcare (1 mentions) Collagenase 4, by Merck Group (1 mentions) Ls003127, by Worthington (1 mentions) Percoll gradient, by GE Healthcare (1 mentions) Papain, by Worthington (1 mentions) Rat anti pdgfrα, by BD (1 mentions) Rat anti cd45, by BD (1 mentions)

3 protocols using ls003086

Isolation of CNS Leukocytes and Oligodendrocytes

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Mice were euthanized through CO₂ asphyxiation and transcardially perfused with ice-cold PBS.
For leukocyte isolation, whole brain and spinal cord was harvested, cut into small pieces and digested with 0.4 mg ml⁻¹ Collagenase IV (9001-12-1, Sigma-Aldrich) and 0.2 mg ml⁻¹ deoxyribonuclease I (DNase I; E1010, Luzerna) in HBSS (with Ca²⁺ and Mg²⁺; 14025-050, Gibco) for 40 min at 37 °C. The digested tissue was then mechanically dissociated using a 19-gauge needle and filtered through a 100-μm cell strainer (800100, Bioswisstec). CNS single-cell suspensions were further enriched by 30% Percoll (P4937, GE Healthcare) gradient centrifugation (1,590g, 30 min, at 4 °C, with no brakes).
To enrich for and improve oligodendrocyte viability for downstream FACS sorting and RNA isolation, CNS tissue was subjected to enzymatic digestion using papain (LS003126, Worthington) for 30 min at 37 °C and the enzymatic reaction stopped with ovomucoid trypsin inhibitor (LS003086, Worthington). Myelin debris was again removed by 30% Percoll (P4937, GE Healthcare) gradient centrifugation.

Andreadou M., Ingelfinger F., De Feo D., Cramer T.L., Tuzlak S., Friebel E., Schreiner B., Eede P., Schneeberger S., Geesdorf M., Ridder F., Welsh C.A., Power L., Kirschenbaum D., Tyagarajan S.K., Greter M., Heppner F.L., Mundt S, & Becher B. (2023). IL-12 sensing in neurons induces neuroprotective CNS tissue adaptation and attenuates neuroinflammation in mice. Nature Neuroscience, 26(10), 1701-1712.

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Isolation of Mouse Brain Cells

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Mice were euthanized using an overdose of ketamine-xylazine, followed by transcardial perfusion with cold PBS, and whole brains were excised. Tissues were chopped into small pieces and subjected to 30 minutes of enzymatic digestion using papain (Worthington, LS003127) in 37°C, followed by manual trituration using a 5ml pipette and filtering through a 70μm cell strainer. Cells were pelleted at 800g for 5 min in 4°C and then suspended in ovomucoid protease inhibitor (Worthington, LS003086) solution to stop papain activity. Cells were pelleted again in 600g, 5 min, 4°C, washed, and myelin debris was removed by 30% Percoll gradient (GE healthcare, 17-0891-01). Then, cells were pelleted and washed again and subjected to 20 min Fc block (1:200), following 30 min cell surface staining. Immediately before FACS, cells were washed, filtered through a 70μm cell strainer, suspended in sorting buffer (PBS supplemented with 0.2mM EDTA pH8 and 0.5% BSA), and DAPI was added. Samples were kept on ice at all times except for enzymatic digestion.

Kenigsbuch M., Bost P., Halevi S., Chang Y., Chen S., Ma Q., Hajbi R., Schwikowski B., Bodenmiller B., Fu H., Schwartz M, & Amit I. (2022). A shared disease-associated oligodendrocyte signature among multiple CNS pathologies. Nature neuroscience, 25(7), 876-886.

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Immunopanning for Cell Type Isolation

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Immunopanning experiment was carried out as previously described with some modifications (Foo, 2013 (link)). The rat or mouse brain was minced and digested with 1 mg/mL papain at 37°C for 1 h. After the cessation of digestion by ovomucoid (Worthington, LS003086), the resulting cell suspension was incubated with primary antibodies [astrocyte, mouse anti-ITGB5 (Invitrogen, 14–0497-82); OPC, rat anti-PDGFRα (BD Biosciences, 558774); OL, mouse anti-PLP (Millipore, MAB388); mouse microglia, rat anti-CD45 (BD, 550539); neuron, rabbit anti-p75 (Abcam, EP1039Y)] diluted in the panning buffer for 30 min at room temperature. The cell suspension was then transferred to a series of secondary antibodies coated 10-cm dishes, and incubated for 30 min. After attachment of the target cell types to the dish, the remnant cell suspension was removed, and the dish was washed with the panning buffer thrice. The cells were then collected with RIPA buffer and subjected to proteomic analysis.

Su Y., Wang X., Yang Y., Chen L., Xia W., Hoi K.K., Li H., Wang Q., Yu G., Chen X., Wang S., Wang Y., Xiao L., Verkhratsky A., Fancy S.P., Yi C, & Niu J. (2022). Astrocyte endfoot formation controls the termination of oligodendrocyte precursor cell perivascular migration during development. Neuron, 111(2), 190-201.e8.

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