Obtaining Cell Suspensions from Ischemic Mouse Brain Tissues

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