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.

In this procedure, using a clean, single-edged razor blade, isolate the area of the brain, containing the tumor by making a sagittal cut down the center of the brain to bisect the two hemispheres. Turn the ipsilateral hemisphere medial side down, and make one coronal cut at the cerebellum, and another cut at the olfactory bulb to isolate the target tissue containing the tumor implant.

Next, place the target tissue into the labeled glass-down tissue grinder, containing 1 milliliter of DPBS. Push the plunger all the way down, and twist seven times to disrupt the tissue. After that, lift the plunger to allow the liquid to settle back to the bottom of the tissue grinder. Repeat this step thrice. However, only twist the plunger four times during the next two repeats and three times during the last repeat to avoid over-triturating the brain tissue.

Subsequently, apply three 1-milliliter volumes of ice-cold DPBS to the sides of the plunger to rinse the residual brain matter into the tissue grinder. Then, resuspend the triturated brain matter by pipetting up and down and place into a labeled 15-milliliter centrifuge tube on ice. Rinse the sides of the DAPS tissue grinder with an additional 1 milliliter of ice-cold DPBS and add to the same 15-milliliter centrifuge tube.

Keep all the tubes containing triturated brain matter on ice, until all the samples have been processed. Then, spin down the triturated brain matter in the 15-milliliter centrifuge tubes at 740 times g for 20 minutes at 4 degrees Celsius. Next, remove the supernatant and resuspend the pelleted brain matter in 1 milliliter of a previously prepared mixture of collagenase and DNase I digestive enzymes.

Place the 15-milliliter centrifuge tubes into a test tube rack, and place the rack in a 37 degrees Celsius water bath for 15 minutes, then, gently agitate the samples twice throughout the incubation period by flicking the tubes to facilitate tissue disaggregation.

Subsequently, add 6 milliliters of ice-cold DPBS to each tube to dilute the digestive enzymes. Pipette up and down to resuspend, and filter the total volume through sterile 70-micron nylon mesh filters into new labeled 15-milliliter centrifuge tubes on ice.

Afterward, spin the brain cell suspension down at 740 times g for 20 minutes at 4 degrees Celsius to achieve a single-cell pellet. Remove the 15-milliliter tubes from the centrifuge and place them on ice. Then, increase the temperature setting on the centrifuge to about 21 degrees Celsius in preparation for the next step.

Next, fully remove the cell supernatant and initially resuspend the cell pellets in 1 milliliter of 70% density centrifugation media, using a P1000 micropipette. Then, add four additional milliliters of 70% density centrifugation media to each tube. Put the caps on securely and homogenize the cell suspension by gently inverting the tube several times.

After that, transfer the 15-milliliter centrifuge tubes, containing brain cells resuspended in 70% density centrifugation media from ice to a test tube back at room temperature. One at a time, carefully overlay 2 milliliters of 37% density centrifugation media solution onto the 5 milliliters of 70% density centrifugation media to form a clean interface between the two density centrifugation media layers.

Then, mark the position of the interface between the two layers with a fine-tipped marker so that it can be easily identified after centrifugation when the distinction becomes less apparent. Spin down the 15-milliliter centrifuge tubes at 740 times g for 20 minutes at room temperature with no break to avoid disrupting the interface between the density centrifugation media layers.

Afterward, collect the PBMCs that have accumulated at the interface between the two density centrifugation media layers by introducing a P200 micropipette into the tube, carefully bypassing the lipid layer. Once at the level of the PBMC band, slowly extract 200 microliters from the surface of the 70% density centrifugation media layer and transfer to a respectively labeled polypropylene FACS tube on ice. Repeat this step once to achieve a total volume of 400 microliters per sample.