Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Take a perfused mouse brain containing a tumor.

The perfusion step removes circulating immune cells but retains the tumor-infiltrated cells.

Isolate the tissue containing the tumor.

Now, mechanically dissociate the tissue.

The mechanical force loosens the tissue, releasing the cells.

Transfer the dissociated tissue to a tube.

Centrifuge and discard the supernatant containing debris.

Resuspend the tissue in a digestion solution containing collagenase and DNase.

Collagenase degrades the extracellular matrix, releasing the cells. DNase degrades contaminating DNA.

Add a buffer to stop the enzymatic activity.

Filter the suspension to remove cell aggregates and obtain a single-cell suspension.

Centrifuge and discard the supernatant.

Resuspend cells in a high-density gradient medium. Now, overlay with a lower-density gradient medium, forming a separation gradient with a clear interface.

Centrifuge to separate layers. Immune cells gather at the interface between gradient mediums while cell debris floats atop.

Collect the immune cells for further analysis.