In this application protocol, we present a protocol to generate highly purified and viable endothelial cells from adult mouse brain tissue. Brain tissue from mice older than P7 is dissociated into single-cell suspensions using the Adult Brain Dissociation Kit and the gentleMACS™ Octo Dissociator with Heaters for mechanical dissociation during on-instrument enzyme incubation. After dissociation, the myelin and cell debris is removed using the Debris Removal Solution and is followed by subsequent removal of erythrocytes using the Red Blood Cell Removal Solution. Endothelial cells are enriched by depletion of CD45+ cells with CD45 MicroBeads followed by a positive selection using CD31 MicroBeads.
|Enzyme mix 1||Enzyme mix 2|
Coat the cell culture dishes with Human Fibronectin (Fragment). The coating concentration should be 3 μg of Human Fibronectin per cm2. For example, if coating a 96-well plate (0.32 cm2 per well):
|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 2 mL tube using 300 µL of Debris Removal Solution, 1000 µL of D-PBS for resuspension of the cell pellet, and ~700 µL D-PBS for overlay.
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|Column||Max. number of labeled cells||Max. number of total cells||Separator|
|MS||1x10⁷||2x10⁷||MiniMACS™, OctoMACS™, |
VarioMACS, SuperMACS II
|LD||2x10⁷||4x10⁷||MidiMACS™, OctoMACS™, |
VarioMACS, SuperMACS II