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.
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).
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:
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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