Isolating Tumor-Infiltrating Immune Cells from a Murine Brain
Trascrizione
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.
