We isolated, expanded, and differentiated OPCs into OLs as previously described (Dincman et al., 2012 (link)). Briefly, we dissociated cells from postnatal day 6 (P6) mouse cortices using the papain-based Neural Tissue Dissociation Kit (Miltenyi) and mechanical trituration, then enriched for OPCs using magnetic cell sorting (MACS) with anti-PDGFRα-conjugated microbeads and a miniMACS separator. OPCs were expanded for 3 days, passaged to new plates, then differentiated for 6 days (or maintained in proliferation media and harvested as OPCs). On the sixth day (D6), cells were mock-treated with media, or treated with a low (0.5 μg/ml) or high (5 μg/ml) dose of morphine sulfate, then harvested for qRT-PCR at 3 h post-treatment. Values shown in Figure S6 are means from three biological replicates.
Papain based neural tissue dissociation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The Papain-based Neural Tissue Dissociation Kit is a laboratory product designed to facilitate the dissociation of neural tissues into single-cell suspensions. The kit contains the enzyme papain and other components necessary for the effective and gentle dissociation of various types of neural tissues, such as brain, spinal cord, or retina.
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Lab products found in correlation
Sh800, by Sony (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Cd16 cd32 monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Hamster igg, by Jackson ImmunoResearch (1 mentions)
Anti fc receptor antibody, by BD (1 mentions)
Facs vantage se diva cell sorter, by BD (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Percoll gradient, by GE Healthcare (1 mentions)
Pe conjugated anti cd45, by BD (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Triiodothyronine t3, by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Pdgf bb, by Merck Group (1 mentions)
Neurobasal, by Merck Group (1 mentions)
L glutamine, by Euroclone (1 mentions)
5 protocols using papain based neural tissue dissociation kit
1
Oligodendrocyte Differentiation and Morphine Effects
2
Isolation and FACS analysis of brain cells
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The mice were anesthetized and perfused with cold PBS. Each brain was removed, and the lesions were microdissected individually under a dissection microscope. Dissociation of single cells was performed by using gentleMACS Dissociator with the Papain-based Neural Tissue Dissociation Kit (Miltenyi Biotec). Cell pellets were blocked with mouse FcR-blocking reagent (1:100, CD16/CD32 monoclonal antibody; eBioscience) and stained for 15 min using 7-AAD (25 μg/ml; Thermo Fisher Scientific) and the antibodies against CD45 (1:200, eFluor450, 30-F11; eBioscience) and CD11b (1:200, PE/Cy7, M1/70; eBioscience) followed by washing with PBS. FACS analysis was performed with flow cytometry (SH800; Sony). Flow cytometry data were analyzed using FlowJo v10.
3
Microglia Isolation and Myelin Phagocytosis
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Microglia were isolated from C57BL/6, P6-P8 WT mice by MACS technology. Brain tissue was dissociated using a Papain-based Neural Tissue Dissociation Kit (Miltenyi Biotec). Briefly, brain tissue was removed, cut into small pieces, and dissociated by enzymatic digestion provided in the kit. The tissue suspension was applied to a 70-μm cell strainer and washed twice with DMEM containing 1 mM sodium pyruvate. The final pellet was resuspended in 10 vol of DMEM containing 10% FCS, 1 mM sodium pyruvate, and 1% antibiotics (DMEM/FCS) plus 1 vol CD11b microbeads (130-093-634; Miltenyi Biotec) and incubated at 4°C for 15 min. After washing with DMEM/FCS, the pellet was resuspended in 500 μl of the same medium and applied to a MACS column in the magnetic field after washing three times; the CD11b+ cells (microglia) were flushed out of the column and centrifuged at 400 ×g for 10 min at 4°C. Isolated microglia were plated on 8-mm coverslips at 7 × 104 cell/ml and incubated until confluent. Myelin was sonicated in an ultrasound water bath for 5 min. Primary microglia cultures were treated with 8 μg myelin and incubated for 2–24 h. The cells were then fixed with 4% PFA and stained for PLIN2, DyLight 694–labeled tomato lectin, and 2 μg/ml Hoechst 33342. The cells were imaged on a Leica SP5 confocal microscope with a 63× objective.
4
Isolation and Characterization of Astrocytes and Microglia
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Brains and spinal cords from transgenic GFAP-EGFP mice were dissociated using the papain-based neural tissue dissociation kit (Miltenyi Biotec, Germany). Myelin was separated from the cells on a discontinuous Percoll gradient (GE Healthcare Biosciences AB, Uppsala, Sweden) and the cells were washed and incubated with blocking solution containing HBSS, FBS (Sigma-Aldrich), anti-Fc receptor antibody (BD Biosciences, Brøndby, Denmark), hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), and sodium azide. The cells were then labeled with phycoerythrin (PE)-conjugated anti-CD45 (BD Biosciences) for 15 min at 4 °C and propidium iodide (PI, Sigma-Aldrich) to detect microglia/macrophages and non-viable cells, respectively.
Cells were sorted using a FACSVantage SE DiVa cell sorter (BD Biosciences). Astrocytes were defined as EGFP positive and CD45 negative (Fig.6 b). Microglia were defined as EGFP negative and CD45dim. Sorted astrocytes and microglia were re-analyzed by flow cytometry and quantitative real-time RT-PCR to verify purity.
Cells were sorted using a FACSVantage SE DiVa cell sorter (BD Biosciences). Astrocytes were defined as EGFP positive and CD45 negative (Fig.
5
Isolation and culture of rat OPCs
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We isolated OPCs from rat (Sprague-Dawley) cerebral cortex at postnatal day 2 (P2) by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) utilizing a Papain-based Neural Tissue Dissociation Kit (Miltenyi Biotec, Bologna, Italy) and anti-A2B5 magnetic microbeads (Miltenyi Biotec, Bologna, Italy). The cells were plated onto poly-d,l-ornithine-coated glass coverslips in Neurobasal (Life Technologies, Monza, Italy) supplemented with 2% B27 (Life Technologies, Monza, Italy), l-glutamine (2 mM; EuroClone, Milan, Italy), human platelet-derived growth factor (PDGFBB, 10 ng/ml; Sigma-Aldrich, Milan, Italy), and human basic fibroblast growth factor (bFGF, 10 ng/ml; Space Import Export, Milan, Italy), to promote proliferation (proliferating medium). 3 days after, we detached OPCs with accutase (Millipore, Burlington, MA, USA) for migration analysis or incubated the cells with a differentiating medium, i.e. Neurobasal medium devoid of growth factors and containing triiodothyronine T3 (10 ng/ml; Sigma Aldrich, Milan, Italy).
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