Isolating Immune Cells from Mouse Choroid Plexuses
Transcripción
To begin, position the brain dissected from a C57 black 6 mouse dorsal side up in a Petri dish, and place the dish below the objectives of the binocular loupes. While maintaining the brain in place using forceps, insert the two ends of another pair of forceps down through the midline between the hemispheres.
Using the forceps, pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle. Identify the lateral choroid plexus as a long veil lining the lateral ventricle and flaring at both ends. Then, using two ends of thin forceps, collect the lateral choroid plexus. Be careful to collect the triangular posterior part hidden by the posterior fold of the hippocampus.
Next, pull the cortex with the corpus callosum and hippocampus of the contralateral hemisphere, away from the septum to expose the entire third ventricle and the opposite lateral ventricle. Using fine forceps, collect the third choroid plexus identified as a short structure with a granular surface aspect. Then, collect the other lateral choroid plexus.
Next, insert the two ends of the forceps down between the cerebellum and midbrain and detach the cerebellum from the pons and medulla to expose the fourth ventricle. Then, identify the fourth choroid plexus as a long, globular structure with a granular surface aspect that lines the fourth ventricle from the lateral right end to the left end between the cerebellum and medulla. Using fine forceps, collect the fourth choroid plexus.
After incubating all collected choroid plexuses with collagenase IV, gently pipette up and down around 10 times to finalize the dissociation. Then, fill the tube to 1.5 milliliters with MACS buffer to stop the collagenase IV activity. Next, centrifuge the cells and discard the supernatant before washing the cells with 1.5 milliliters of MACS buffer. After adding the appropriate compensation beads and antibody solutions, incubate the samples on ice for 30 to 45 minutes, protecting them from light exposure. Then, fill the tubes with 1.5 milliliters of MACS buffer.
