Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Low cellular viability and the larger cell debris are the main difficulties of vascular tissue dissociation. Our two-step cell digestion method is very suitable for carotid tissue dissociation and may be used to prepare single-cell suspensions for other vessels with minor modifications, making it very helpful in cardiovascular research. This protocol is suitable for obtaining multiple cell types in the carotid artery, and it can be easily repeated without special equipment.

In the future, we aim to use cell subculture discovered by single-cell iron sequencing for targeted therapy of cardiovascular disease. To begin, spray the euthanized mouse with 75%alcohol. Then, use sterilized dissecting scissors to open the skin and expose the chest cavity.

Cut the diaphragm. Then, continue cutting upward toward the lower jaw and carefully remove excess connective tissues and fat, exposing the carotid artery. Next, cut the inferior vena cava to make blood flow out of the closed circulation.

Using a syringe, slowly inject 20 milliliters of pre-chilled perfusion solution into the left ventricle. Place the mouse under a stereoscopic microscope, then, using micro dissecting scissors, peel off the fat and connective tissues around the carotid artery. Isolate the carotid artery and wash it with PBS to flush away any residual blood.

Transfer the artery to a six-well plate containing 1.5%FBS on ice. Using micro dissecting scissors, dissect the carotid artery longitudinally and place it flat in a new six-well plate containing one milliliter of FBS on ice. Carefully flush the intima of the artery with FBS to remove the residual blood.

Cut the artery into two millimeter square tissue pieces and transfer them into a 1.5 milliliter centrifuge tube. Centrifuge at 400 G for five minutes at four degrees Celsius to sink the tissues to the bottom. Discard the supernatant and resuspend the tissues in 500 microliters of dissociation reagent A.Place the tube in a 37 degree Celsius water bath for one hour, pipetting gently with a one milliliter pipette every 10 minutes.

Then, add 500 microliters of 5%FBS into the tube and mix well. Filter the cell suspension through a sterile 40 micron cell strainer into a 1.5 milliliter tube, then centrifuge the filtrate and remove the supernatant before resuspending the pellet in 200 microliters of dissociation reagent B.Place the tube in a water bath to fully dissociate cells into a single-cell suspension. After five minutes, add 200 microliters of 5%FBS to terminate the digestion reaction.

Centrifuge the suspension and discard the supernatant before resuspending the pellet in 100 microliters of PBS on ice. Microscopic observations showed that combining dissociation reagent B with reagent A led to more thorough dissociation of carotid artery tissue compared to using dissociation reagent A alone. A total of 176, 000 single cells were collected from nine left carotids, with most vascular vessels being successfully dissociated into single cells with high viability.

After digestion, unsupervised CIARA-based clustering revealed four main cell types in normal mouse carotid arteries.