In this application protocol, we generate highly purified and viable microglia from adult mouse or rat brain tissue. Brain tissue from mice or rats older than P7 is dissociated into single-cell suspensions using the Adult Brain Dissociation Kit. The extracellular matrix is enzymatically digested using the kit components, while the gentleMACS™ Dissociator with Heaters is used for the mechanical dissociation steps during the on-instrument enzyme incubation. After the dissociation, the myelin and cell debris are removed using the Debris Removal Solution and is followed by subsequent removal of erythrocytes using the Red Blood Cell Removal Solution. The CD11b (Microglia) MicroBeads, mouse and human or the CD11b/c (Microglia) MicroBeads, rat are used to isolate microglia from the single-cell suspension.
The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
|Column||Max. number of labeled cells||Max. number of total cells||Selector|
|MS||1 x10⁷||2 x10⁷||MiniMACS™, OctoMACS™,|
VarioMACS, SuperMACS II
|LD||2 x10⁷||4 x10⁷||MidiMACS™, QuadroMACS™,|
VarioMACS, SuperMACS II
|autoMACS||5 x10⁷||1 x108||autoMACS Pro|
|Enzyme mix 1||Enzyme mix 2|
|Debris Removal Solution||D-PBS||Overlay (D-PBS)|
|900 µL||3100 µL||4 mL|
|1800 µL||6200 µL||4 mL|
▲ Note: In case of very small amount of tissue (< 100 mg) cell debris removal can be performed in a 5 mL reagent tube using 450 µL of Debris Removal Solution, 1550 µL of D-PBS for resuspension of the cell pellet, and 2000 µL D-PBS for overlay.
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
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