Dissected embryonic E14.5 brains were titrated in FACS buffer (PBS - 1% Fetal calf serum - 0.02% NaN3) using pipette tip, and filtered through 70-µm cell strainers (Fisher Scientific, Hampton, NH). Single cell suspensions were blocked with 10% normal rat serum on ice for 30 min., and stained with a combination of anti-mouse cell surface flow cytometric markers CD11b-FITC (BioLegend, San Diego, CA), GLAST-PE, and O4-APC (Miltenyi Biotec, Bergish Gladbach, Germany) at 4 °C in dark for 1 h with continuous rotation. The stained cells were then washed, centrifuged, and resuspended into 1 mL of FACS buffer, and acquired on a two-laser, six-color Gallios cytometer (Beckman Coulter, Pasadena, CA). The threshold for FACS analysis was set based on the surface expression of each individual marker in the samples as compared to isotype control using IgG-stained cells in the same channel, and Flow cytometric data were analyzed with the Kaluza 1.3 software (Beckman Coulter, Brea, CA), as reported [35 (link),36 (link)]. Cell populations were calculated as the percentages among the total cells.
O4 apc
Manufactured by Miltenyi Biotec
Sourced in Spain, United States
The O4-APC is a monoclonal antibody conjugated with the fluorescent dye allophycocyanin (APC). It is designed for the detection and analysis of O4-positive cells, which are typically found in the oligodendrocyte lineage. This product can be used in flow cytometry applications to identify and quantify O4-positive cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Acsa 2 pe, by Miltenyi Biotec (3 mentions)
Adult brain dissociation kit, by Miltenyi Biotec (2 mentions)
Cd11b fitc, by BioLegend (1 mentions)
70 m cell strainer, by Thermo Fisher Scientific (1 mentions)
Kaluza 1, by Beckman Coulter (1 mentions)
Glast pe, by Miltenyi Biotec (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Cd11b vio bright fitc, by Miltenyi Biotec (1 mentions)
Facsverse cytometer, by FlowJo (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
Mouse fcγ receptor block reagent, by BD (1 mentions)
Apc cy7 rat anti cd11b clone m1 70, by BD (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Cyto expert software, by BD (1 mentions)
Pdgfra apc, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd11b pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd45 fitc, by BioLegend (1 mentions)
Gentlemacs octo dissociator with heater, by Miltenyi Biotec (1 mentions)
Neuron isolation kit, by Miltenyi Biotec (1 mentions)
6 protocols using o4 apc
1
Embryonic Brain Cell Isolation and Analysis
2
Astrocyte Purity Validation by Flow Cytometry
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The purity of the astrocyte cultures was confirmed by flow cytometry. First, cells were blocked with FcR blocking reagent (#130-092-575, Miltenyi Biotec, Madrid, Spain,) in 1× phosphate buffer saline (PBS) and 0.5% bovine serum albumin (BSA) and then stained with combinations of fluorophore-conjugated antibodies purchased from Miltenyi Biotec: for astrocytes, ACSA-2-PE (#130-102-365); for microglia, CD11b-Vio Bright FITC (#130-109-368); and for oligodendrocytes, O4-APC (#130-095-895). Data were acquired on a FACSVERSE cytometer and analyzed using FlowJo v10 software (Ashland, OR, USA). Non-viable cells were excluded based on forward and side scatter profiles and 7-Aminodactinomycin (7AAD) staining (#130-111-568).
3
Single-Cell Dissociation and Sorting
4
Characterizing OLIG2 Progenitor Cells
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Cells were enzymatically harvested using Accutase, centrifuged and resuspended in 100 μL of FACS buffer (1X PBS, 2% fetal bovine serum and 0.02% sodium azide). To determine NG2, PDGFRa, and O4 expression, OLIG2 smRNA-induced progenies were incubated or not (as a control) with 1/20 dilutions of NG2-PE (C06035P, Signalway antibody, USA), PDGFRa-APC (323512, BioLegend, USA), and O4-APC (130-118-978, Miltenyi Biotec, Germany) antibodies or for 15 min at 4 °C, washed and resuspended in 150 μL of FACS buffer. The cells were analyzed using a BD flow cytometer (Beckman Coulter, CytoFLEXS). All flow cytometry data were further analyzed using BD CytoExpert software (BD Biosciences, USA).
5
Neuronal Isolation from Adult Mouse Brains
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Whole brains from adult female 10- to 15-wk-old mice were dissociated using the Adult Brain Dissociation Kit and Gentle Macs Octo Dissociator with Heaters (Miltenyi) according to the manufacturer’s protocol. Non-neuronal cells were removed using the Neuron Isolation Kit (Miltenyi), which contains biotinylated antibodies against microglia, oligodendrocytes, and astrocytes, conjugated to magnetic microbeads that are removed by magnetic-activated cell sorting (MACS). The flow-through containing highly enriched neuronal cells was collected. The purity of neuronal isolates was assessed by flow cytometry using a FACS Canto II (BD Biosciences) according to the manufacturer’s instructions. In brief, living cells were gated for single cells only (side scatter and forward scatter), and the samples were stained for cell populations using specific antibodies for microglia/macrophages (CD11b PeCy7; Invitrogen), oligodendrocytes (O4 APC; Miltenyi), endothelial cells (CD31 PEVio770; Miltenyi), thrombocytes/immune cells (CD45 FITC; Biolegend), and astrocytes (astrocyte cell surface antigen-2 [ACSA-2] PE, Miltenyi; Table 1 ). Neuronal purity of 90–96% was achieved.
6
Single-Cell Sequencing of Specific Cell Types
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For single-cell sequencing experiments, single-cell suspensions were stained for 30 min at room temperature with the following antibodies: O4-APC (O4, 10 µL/test, Miltenyi, 130-095-891), CD11b-e450 (M1/70, 0.5 µL/test, eBioscience, 48-0112-82), TER119-APC/Cy7 (TER-119, 1.25 µL/test, Biolegend, 116223), PDGFRα-PE/Cy7 (APA5, 0.625 µL/test, Invitrogen, 25-1401-82), CD45-PerCP/Cy5.5 (30-F11, 0.5 µL/test, eBioscience, 45-0451-82), and CD16/31 (93, 0.5 µL/test, Invitrogen, 14-0161-82). Viability was determined using Ghost Dye Violet 510 (0.5 µL/test, Tonbo biosciences, 13-0870). Cells were sorted using a 16-color BD influx cell sorter. Cells used for sequencing were gated on live/singlets/TER119−/CD45−/CD11b−/YFP+. Following sorting, cells were washed three times with 0.04% BSA and then processed for sequencing according to the 10× Genomics protocol.
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