In a blood vessel, the number of endothelial cells is very low, which makes it difficult to study them. Our protocol eliminates this limitation, and allows us to study novel molecular pathways and therapeutics. Our protocol allows us to obtain endothelial enriched samples, and study how acute and chronic disturbed flow affects this cell type.
The ability to study endothelial cells in more detail makes it possible to define target genes, and therefore potential therapeutics for atherosclerosis targeting endothelial cells. Carotid artery isolation is tricky, and requires patience and experience. To begin, prepare the animal by injecting 0.05 milligrams per kilogram of buprenorphine subcutaneously.
Sterilize the epilated area using Betadine and isopropanol solution three times. Make a ventral midline incision of approximately one centimeter in the neck region. Then expose the LCA branch point by blunt dissection.
Ligate the left internal carotid, external carotid, and occipital arteries with 6-0 sutures, leaving the superior thyroid artery untouched. Close the incision with tissue glue or sutures. Transfer the mouse to a pre-warmed recovery cage kept at 37 degrees Celsius.
Place the mouse on a clean towel for up to one hour to avoid post-surgery hypothermia. Insert a 21 gauge needle into the IV line connected through the apex of the heart into the left ventricle. Allow retrograde perfusion for two to three minutes using normal saline at room temperature.
Maintain a constant flow rate by applying steady pressure while injecting or using an IV line, and keeping the saline bag at a height of eight to nine feet. Remove the skin from the neck region with all the fat, muscles, and connective tissues to expose the carotid arteries. Then remove the periadventitial tissues around the carotids leaving the carotid attached to the body.
Make an incision below the ligation site in the LCA to allow perfusion. Perfuse the LCA through the left ventricle for another minute to remove any blood traces. Wash the exterior of the carotid arteries with normal saline solution to remove any traces of blood.
Use an insulin syringe with a 29 gauge needle to inject 50 microliters of digestion buffer into the lumen of the distal end of the left carotid artery. Use a micro clip to clamp the proximal end of the carotid artery filled with digestion buffer. Add 15 to 20 microliters of digestion buffer into the lumen, and clip the distal end to avoid any release of digestion buffer.
Transfer the carotid arteries from the mouse into 35 millimeter dishes containing warm 37 degree Celsius HEPES Buffered Saline Solution. And incubate them for 45 minutes at 37 degrees Celsius with intermittent rocking. Remove the carotid artery with the clamps from the 35 millimeter dish after the luminal enzymatic digestion is completed.
Detach the clamps carefully, so that the digestion buffer does not leak. Hold one end of the carotid artery on top of a 1.5 milliliter micro centrifuge tube. Add 100 microliters of warm digestion buffer to the lumen by inserting a 29 gauge needle equipped with an insulin syringe.
Quickly flush the lumen of the carotid artery in the micro centrifuge tube, then add 0.3 microliters of FBS into the tube to stop the reaction. Place the micro centrifuge tube on ice. Centrifuge the cells at 500 G for five minutes at four degrees Celsius, using a centrifuge equipped with a fixed angle rotor and discard the supernatant.
Re-suspend the cells in digestion buffer, containing cell dissociation reagent, and incubate them for five minutes at 37 degrees Celsius to separate them into single cells. Block the enzymatic reaction by adding 0.15 milliliter of FBS to the 0.5 milliliter tube, and repeat the centrifugation. Discard the supernatant, and resuspend the cells in 100 microliters of ice cold 1%BSA solution in PBS in a 0.2 milliliter micro centrifuge tube.
For single cell analysis, resuspend the pellet with 100 microliters of ice cold 1%BSA in PBS in a 0.2 milliliter tube. For single nucleus analysis, resuspend the cell pellet in 100 microliters of 0.04%BSA in ice cold PBS, and centrifuge the single cell suspension at 500G at four degrees Celsius for five minutes. Decompose the single cell suspension with ice cold lysis buffer, and incubate for five minutes on ice.
Mix the lysate with a P20 pipette, and incubate for another 10 minutes. Afterward, transfer the lysate in a 0.5 milliliter tube. Wash the ly cells with 500 microliters of buffer, and mix them using a pipette.
Then repeat the centrifugation. Discard the supernatant, and resuspend the nuclei pellet in 150 microliters of the diluted nuclei buffer. Count the single nuclei preparations using a hemocytometer.
Following the single nuclei sequencing study, single cell and single nuclei were obtained and visualized by bright-field, and phase contrast microscopy. The efficiency of single nuclei preparation from the single cell suspension was also demonstrated. After the single cell RNA sequencing study, various parameters such as distribution of genes per cell, unique molecular identifier per cell, mitochondrial reads per cell, and sequencing saturation data were obtained.
For the single cell ATAC sequence study, the insert size distribution, including the nucleosome banding pattern was observed. The normalized TSS enrichment score was also observed. In addition, the percent fragment reads in the peaks.
Peak region fragments, TSS enrichment score, ratio of reads in the blacklist genomic sites, and nucleosome signal ratio were determined. The enzymatic digestion is a stress for the cells, therefore it is crucial to maintain the samples on ice, and perform the procedure as quickly as possible. We can use this method to obtain and plate endothelial cells from mice.
We can also use this technique to perform bulk Omix based studies. This technique paved way for studying the effect of disturbed blood flow on endothelial cells in vivo in a single cell manner.
