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In this application protocol, we describe a protocol to generate highly purified and viable astrocytes from neonatal mouse brain tissue. Brain tissue from mice younger than P8 is dissociated into single-cell suspensions and astrocytes are then isolated using the Anti-ACSA-2 MicroBead Kit. The ACSA-2 antigen is expressed specifically on astrocytes in a pattern similar to GLAST, and serves as a specific marker for astrocytes in the central nervous system.1,2 The proportion of ACSA-2+ astrocytes in brain tissue differs according to mouse age and brain region used for cell isolation.
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PB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution 1:20 with PBS. Keep buffer cold (2−8 °C). Prepare fresh on the day of use and degas the buffer, as air bubbles could block the column.
▲ Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).
Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.
Prepare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.
Use the Neural Tissue Dissociation Kit (P) or Neural Tissue Dissociation Kit (T) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.
Download data sheet
Isolate the ACSA-2-positive astrocytes from the single-cell suspension using the Anti-ACSA-2 MicroBead Kit, mouse. Follow the protocol of the kit data sheet.
Download data sheet
Notes:
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autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec B.V. & Co. KG and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec B.V. & Co. KG and/or its affiliates. All rights reserved.
Column | Max. number of labeled cells | Max. number of total cells | Separator |
---|---|---|---|
Positive selection | |||
MS | 1×10⁷ | 2×10⁷ | MiniMACS™, OctoMACS™, VarioMACS, SuperMACS II |
LS | 2×10⁷ | 4×10⁷ | MidiMACS™, QuadroMACS, VarioMACS, SuperMACS II |
Depletion | |||
LD | 1.5×10⁷ | 3×10⁷ | MidiMACS, QuadroMACS, VarioMACS, SuperMACS II |
Positive selection or depletion | |||
autoMACS | 5×10⁷ | 1×108 | autoMACS Pro |
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