This article describes a detailed protocol for isolating brain cells from perfused ischemic mouse brain tissues. The method involves several steps including tissue mincing, enzymatic digestion, and density gradient centrifugation to obtain a pure cell suspension for further analysis.
Take perfused ischemic mouse brain tissues containing brain cells and infiltrated immune cells.
Mince the tissues on a strainer and rinse with buffer.
Centrifuge the filtrate and discard the supernatant.
Resuspend the pellet in a digestive enzyme solution and transfer it to a microcentrifuge tube.
Incubate for the digestive enzymes to break down the extracellular matrix, loosening the cells.
Filter the suspension and rinse with buffer containing DNase to remove contaminating DNA.
Next, wash with DNase-free buffer.
Centrifuge the filtrate and discard the supernatant.
Resuspend the cells in density gradient medium. Then, transfer the contents to a fresh tube and mix. Centrifuge to separate the components by density.
Remove the supernatant containing myelin and debris.
Resuspend the pellet in DNase-free buffer. Transfer it to a new tube and centrifuge again.
Discard the supernatant to ensure sufficient removal of the density gradient medium.
Finally, resuspend the cells in buffer for further analysis.
Using the plunger end of a 5-milliliter syringe, mash the pieces of brain tissue through a 100-micron cell strainer, continuously rinsing the strainer with ice cold HBSS with calcium and magnesium. Then place the homogenized tissues on ice.
When all of the samples have been processed, spin down the cell suspensions. And carefully discard the supernatants. Resuspend the pellets in 1 milliliter of digestion buffer. Then transfer the cell suspension into a 2-milliliter tube.
And incubate the cells under slow continuous rotation at 37 degrees Celsius. After one hour, sieve the cell suspension through a 70-micron cell strainer. And rinse the filter thoroughly with 3 milliliters of wash buffer containing DNase. Then wash the strainer with 15 milliliters of DNase-free wash buffer. Spin down the cells, and discard the supernatant.
To remove the cell debris and myelin, resuspend the cells in 5 milliliters of room temperature 25% density gradient medium and transfer the cell suspension into a 15 milliliter tube. Mix the cells and gradient thoroughly with repeated gentle pipetting. Then spin down the cell solution.
At the end of the separation, carefully aspirate the myelin coat and supernatant without disturbing the pellet. And resuspend the cells in 10 milliliters of DNase-free wash buffer. Transfer the cells into a 15-milliliter tube, and spin them down again, resuspending the pellet in 100 microliters of cold wash buffer.
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