Isolating Immune Cells from Mouse Brain and Skull

Our research focuses on the complex immune response following brain ischemia caused by ischemic stroke and the cardiac arrest. Brain ischemia triggers the activation resident immune cells in the brain and the infiltration of immune cells. However, our understanding of this immune cells remains limited.

We aim to dissect the immune state origin, and functional roles of this brain cells after ischemia. Recent research highlights the surprising role of the skull in the brain's immune response following injury. Therefore, it is important to analyze changes in immune cells in both the brain tissue and the skull bone marrow after brain ischemia.

To this end, a critical step is to obtain a sufficient number of high quality immune cells for further analysis. Our protocols enable researchers to rapidly obtain large quantities available immune cells from the brain and the skull bone marrow. The brain immune cell protocol uses a mechanical dissociation approach at low temperatures throughout ensuring better preservation of the transcriptional and the proteomic profiles.

The skull bone marrow protocol is simple, yet effective for extracting bone marrow cells from the skull. We'll continue our comprehensive research on the impact of brain ischemia on immune cells in brain tissue, and the skull. Given that neuroinflammation plays a key role in the past physiology of brain injury after ischemia, our research is expected to discover novel therapeutic targets for brain protection after ischemic stroke or cardiac arrest.

To obtain single cell suspension from the isolated murine brain, place the brain in a pre-cool 15 milliliter glass downs Homogenizer containing an appropriate amount of ice cold Hanks balanced salt solution or HBSS buffer. Use the loose downs pestle to gently and slowly dissociate the brain tissue with around 100 to 120 strokes on ice. Filter the resulting brain tissue suspension through a 70 micron cell strainer in a 50 milliliter centrifuge tube.

Rinse the downs tube with five milliliters of HBSS and proceed with filtration to maximize the cell collection Centrifuge the filtered tissue suspension at 550 G for six minutes at four degrees Celsius. Remove the supernatant using a vacuum aspirator and resuspend the cell palate in one milliliter of 30%isotonic density gradient solution. Transfer the cell suspension into a 15 milliliter centrifuge tube.

Add an appropriate amount of 30%density gradient solution and gently invert the tube to ensure thorough mixing. Immediately centrifuge the tube at 840 5G with acceleration and brake set at level three for 20 minutes at four degrees Celsius. Once done, gently remove the tube from the centrifuge without shaking and carefully aspirate the upper mylin layer using a one milliliter tip connected to the vacuum, then aspirate the supernatant, leaving one to two milliliters behind and resuspend the cell palette with a minimum of 10 milliliters cold HBSS.

Centrifuge at 550 G for six minutes at four degrees Celsius. Wash one more time with a minimum of seven milliliters of cold HBSS and centrifuge again after removing as much of the supernatant as possible, resuspend the cells in 100 to 200 microliters of flow cytometry buffer for flow cytometry analysis. To prepare bone marrow single cell suspension from mouse calveria, under a dissecting microscope, carefully peel off the dura mater from the isolated skull using blunt forceps.

Place the calveria in a dish containing PBS kept on ice and use a one milliliter pipette to rinse it ensuring the removal of blood residues from its surface transfer the calveria to a new dish containing enough fresh cold PBS sufficient to immerse it. Use sterile scissors to cut the calveria into approximately three millimeter by three millimeter fragments in cold PBS. Using an 18 gauge needle, poke a hole in the bottom of a 500 microliter micro centrifuge tube.

Insert this tube into a 1.5 milliliter micro centrifuge tube containing 20 microliters of cold PBS. Then transfer the calveria fragments into the 500 microliter tube. Centrifuge the setup at 10, 000 G for 30 seconds at four degrees Celsius.

Resuspend the collected cal bone marrow cells in 500 microliters of red blood cell lysis buffer, and incubate at room temperature for 30 seconds, transfer the suspension to a 15 milliliter centrifuge tube containing four milliliters of cold PBS centrifuge at 400 G for six minutes at four degrees Celsius. Wash one more time with five milliliters of cold PBS. Finally, resuspend the cell palate in an appropriate buffer.