A2B5-positive OPCs were isolated from cortices of C57BL/6J P5 mouse pups by magnetic cell sorting (Miltenyi Biotec), as previously described (Montagne et al., 2018 (link)). Brains were removed, minced, and processed using Neural Dissociation Kit (130-092-628; Miltenyi Biotec). After tissue digestion and dissociation, cells were filtered, centrifuged, and resuspended in DMEM (Invitrogen) containing 1% FBS (Hyclone), incubated with blocking reagent and magnetically labeled anti-A2B5 microbeads (130-093-392; Miltenyi Biotec), followed by magnetic sorting using magnetic cell sorting LS columns (130-042-401; Miltenyi Biotec). OPCs were plated at 5 × 104 cell density on poly-D-lysine–coated coverslips and allowed to differentiate into mature OLs in medium containing DMEM/Sato supplement–based growth medium, Forskolin, NT3, and T3 (T3 is the active hormone; 3,5,30-tri-iodothyronine; Montagne et al., 2018 (link); Zuchero et al., 2015 (link)). Matured OLs were used for OGD and 3K3A-APC experiments.
Anti a2b5 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-A2B5 microbeads are magnetic beads coated with antibodies specific for the A2B5 antigen. They are designed for the isolation and enrichment of A2B5-positive cells from complex samples, such as neural cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Magnetic cell sorting, by Miltenyi Biotec (1 mentions)
Neural dissociation kit, by Miltenyi Biotec (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by Miltenyi Biotec (1 mentions)
Neurobrew, by Miltenyi Biotec (1 mentions)
Pdgf bb, by R&D Systems (1 mentions)
Macs multistand, by Miltenyi Biotec (1 mentions)
Ms column, by Miltenyi Biotec (1 mentions)
B27 supplement, by Merck Group (1 mentions)
Pdgf aa, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
4 protocols using anti a2b5 microbeads
1
Isolation and Differentiation of Oligodendrocyte Progenitor Cells
2
Cultivation of HEK293 and Primary Neural Cells
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HEK293 cells and stable HEK(pTRAFNrf2/HIF/NFkB) reporter cells were cultured in Eagle’s minimum essential medium, supplemented with 10% fetal bovine serum and 100 U penicillin/ml and 100 μg streptomycin/ml. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 and 21% O2. Primary rat cultures were established from neonatal pups. Microglia and oligodendrocytes were isolated as described for flow cytometry; following Percoll layering, cells were labeled with either anti-cd11b or anti-A2B5 Microbeads (Milteny Biotec, Bergisch, D). Microglia were cultured in DMEM/F12 supplemented with 100 U penicillin/ml and 100 μg streptomycin/ml and 5% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA). Oligodendrocytes were cultured in in 200 mL Neurobrew, 100 mL N2-supplement (Milteny Biotec, Bergisch, D), 2 mL bFGF (PeproTech, Princeton, NJ), and 2 mL PDGF-BB (R&D Systems, Minneapolis, MA) per 10 mL of DMEM/F-12 media on poly-l -lysine pre-coated culture ware. For differentiation of oligodendrocytes, bFGF and PDGF-BB was removed 48 h prior to stimulation.
3
A2B5+ Cell Enrichment Protocol
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A2B5 positive selection was performed following manufacturer´s instructions with anti-A2B5 MicroBeads (human, mouse, rat; #130-093-392) on a MACS® MultiStand (#130-042-303) using MS Columns (#130-042-201), all from Miltenyi Biotec, Germany. Prior to magnetic separation, tumor cell suspensions were treated with 20 µl of FcR Blocking Reagent (#130-059-901) for 10 min and subsequently with 20 µl of anti-A2B5 MicroBeads for 15 min. After magnetic separation, cells (from both fractions, positive-selected and flow through, respectively) were centrifuged at 300×g for 10 min, immediately resuspended in culture medium (ABM or NSM) and transferred to a 37.0 °C incubator with humidified atmosphere and 5% CO2.
4
Oligodendrocyte and Astrocyte Differentiation from A2B5+ Cells
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Enriched populations of A2B5 + cells were isolated from the brains of C57BL/6J mice at postnatal day 5 via magnetic activated cell sorting with anti-A2B5 microbeads (Miltenyi Biotec, 130-093-392) according to the manufacturer’s instructions. The cells were plated on PLL-coated coverslips and differentiated into O4 + oligodendrocytes or GFAP + astrocytes. To promote OPC proliferation, A2B5 + cells were initially seeded at 10,000 cells per well and cultured for 24 h in medium supplemented with DMEM/F12 and 2X B-27 supplement (Thermo Fisher, 12587010), 2 mM l -glutamine (Gibco, 25030081), 20 ng/mL PDGF-AA and 20 ng/mL bFGF (PeproTech, 100-18B). The cells were then switched to oligodendrocyte differentiation medium containing DMEM/F12 supplemented with 2X B-27 supplement, 2 mM l -glutamine, 10 ng/mL PDGF, 30 ng/mL T3 (Sigma‒Aldrich, T63-97), and 10 ng/mL NT-3 (PeproTech, 450-03). Cultures were differentiated for 2 or 6 days before treatment with 10 µL of Nef or Ctrl EVs for 48 h and then analyzed via immunohistochemistry. To promote astrocyte differentiation and proliferation, A2B5 + cells were initially seeded at 10,000 cells per well and cultured in “astrocyte media”, DMEM/F-12 supplemented with 5% FBS, 1% penicillin‒streptomycin, N2, and 10 ng/mL PDGF-AA. After 3 days, the cells were treated with 10 µL of Nef or Ctrl EVs for 48 h and then analyzed via immunohistochemistry.
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