Isolation of Pericytes from Mouse Cortical Tissue Using FACS for Single-cell Sequencing

Our research examine brain parasites focusing on their vascular roles and their interactions within the neurovascular unit to understand the cerebral function. Low imaging and the spatial FACS in a bowel process mapping or parasite function within brain vasculature revealing the location specific roles and therapeutic targets. To begin, spray the head and neck region of a euthanized mouse with 75%ethanol.

Using a scalpel, make a midline scalp incision from the occipital to the frontal region to expose the skull. Perform a transverse incision anterior to the bilateral orbits, followed by lateral incisions along the inferior margins of the cerebellum to detach the skull base. Incise the calvarium longitudinally along the sagittal suture from posterior to anterior.

Lift the cranial flap together with a duramater to expose the brain. Using surgical scissors, gently elevate the brain while severing the optic nerves and the medulla oblongata spinal cord connection. Place the intact brain gently into a 10 centimeter dish containing 10 milliliters of ice-cold PBS, avoiding any damage.

Transfer the brain onto aluminum foil. Expose the subarachnoid space. After removing the dura with forceps, rinse it with Hank's balanced salt solution.

Transfer the tissue to filter paper and remove the piamater by rolling. With a razor blade, cut and discard the olfactory bulb, and the caudal most sections, and section the brain coronally into slices two to three millimeters thick. Isolate the cerebral cortex along the corpus callosum.

Transfer the cortical tissue into ampules containing three milliliters of ice cold tissue collection buffer. Using surgical scalpels, mince the tissue and ampules into fragments of approximately one by one millimeter, and complete the mincing within five minutes to preserve tissue freshness. Pre-rinse pipette tips with tissue collection buffer.

Then slowly aspirate the minced fragments toward the tip apex while holding the pipette vertically. Gently expel the fragments into pre-chilled 15 milliliter centrifuge tubes. Wash the original ampules with 2%FBS in PBS saline.

Transfer the wash into the same centrifuge tubes and centrifuge at 300G for five minutes at four degrees Celsius. Discard the supernatant after centrifugation. Re-suspend each pellet in three milliliters of CD enzyme working solution.

Aliquot the suspension equally into three tubes, and add additional enzyme solution to reach a final volume of five milliliters per tube. Incubate the tubes in a hybridization oven at 37 degrees Celsius for 100 minutes with orbital shaking at 100G. During incubation, add one milliliter of dnase one solution to 10 milliliters of 2%FBS in PBS.

Terminate digestion by centrifuging at 300G at four degrees Celsius for five minutes. Then re-suspend each pellet in one milliliter of tissue dissociation solution. Pipette the solution 100 times to aid mechanical dissociation.

Next, pool all suspensions into a single 50 milliliter centrifuge tube. Rinse each tube with two milliliters of buffer and combine the washes. After centrifugation at 300G for five minutes at four degrees Celsius and discarding the uppermost layer, add 12.5 milliliters of 20%BSA and invert to mix.

Centrifuge at 1000 G at four degrees Celsius for 20 minutes. Now collect the bottom pellet and transfer the supernatant into a new 50 milliliter centrifuge tube. Re-suspend gently before centrifuging again.

Re-suspend each pellet in two milliliters of 2%FBS in PBS. Then combine both suspensions into a single tube. Centrifuge the combined suspension at 300G at four degrees Celsius for five minutes.

After discarding the supernatant, re-suspend the final pellet in two milliliters of PBS with 2%FBS. Prepare four 15 milliliter centrifuge tubes containing four milliliters of 22%per call solution. Gently layer 0.5 milliliter aliquots of the cell suspension onto each gradient and centrifuge at 560G for 10 minutes at four degrees Celsius.

After aspirating the supernatant, re-suspend each pellet in one milliliter of HBSS BSA glucose buffer. Then pull the suspensions into a new 15 milliliter tube. Rinse the percol tubes with two milliliters of buffer and combine the washes before centrifugation at 300G for five minutes at four degrees Celsius.

For flow cytometry, re-suspend the pellet in one milliliter of HBSS BSA glucose buffer after aspirating the supernadant. Add five microliters of FC receptor blocking reagent and incubate at four degrees Celsius for five minutes. Then pipette fluorescently conjugated antibodies to the suspension before incubating.

Wash the cells with six milliliters of HBSS BSA glucose buffer. Then centrifuge the suspension at 300G at four degrees Celsius for five minutes. After aspirating the supernatant, re-suspend the pellet in one milliliter of buffer.

Add 200 microliters of seven amino actinomycin D solution and incubate on ice for 10 minutes. Then adjust the final volume to two milliliters with HBSS buffer supplemented with BSA and glucose. Rinse the inner surface of sorting collection tubes with 1%BSA solution.

Aspirate out the excess solution, leaving exactly one milliliter of solution in each tube. Store the tubes on ice to receive sorted cells. Cells dissociated from brain tissue were sequentially gated by forward scatter area and side scatter area to exclude cellular debris during flow cytometry sorting.

CD45 positive and CD41 positive dual positive events were excluded to remove hematopoietic cells and platelet contaminants. CD13 positive vascular wall cells were enriched. And apoptotic cells were excluded using seven amino actinomycin D negative gating, followed by CD31 negative selection to isolate parasites.

Flow cytometry sorting showed that nucleated cells accounted for 59.8%of all events. CD13 positive cells accounted for 1%of all events. And the final CD13 positive CD31 negative parasite population accounted for 0.7%of all events.

UMAP dimensionality reduction of the sorted cells revealed six distinct major clusters indicating cellular heterogeneity within the isolated population. Z-score normalized heat map analysis demonstrated that parasite marker genes were highly and predominantly expressed in clusters zero through three. Cluster proportion analysis showed that clusters zero through three constituted 87.1%of the total population.

Whereas smooth muscle cell markers were enriched in cluster four at 10.14%and fibroblast markers were enriched in cluster five at 2.77%This protocol allows researchers to isolate high purity wearable parasites suitable for downstream single cell sequencing and functional analysis. The main challenge is the absence of highly specific parasite markers, complicated isolation, and increasing the risk of secular contamination. Future studies can focus on identifying parasite specific markers, enabling improved isolate, deeper functional characterization, and targeted therapeutic investigations.