Protocol for Single-Cell Dissociation, RNA Sequencing, and Isolation of Myoepithelial Cells and Fibroblasts from the Aged Lacrimal and Salivary Glands

My lab studies how age-related changes in salivary and lacrimal glands affect different cell types using flow cytometry and single-cell RNA sequencing. Standard dissociation methods poorly recover fragile epithelial cells from aged salivary and lacrimal glands. And my protocol improves cell viability and enable reliable single-cell analysis and cell sorting.

To begin, position the euthanized mouse supine on the dissection table. Using fine forceps and scissors, incise the skin between the eye and the ear. Gently retract the skin to expose the lacrimal gland.

Using dull-tipped forceps, lift the lacrimal gland slightly. Carefully separate the surrounding connective tissue by gently teasing it away in a circular motion. Identify the visible boundaries of the lacrimal gland and separate it from adjacent tissues, including the parotid gland.

Then transfer the lacrimal gland to a 35-millimeter Petri dish containing two milliliters of cold PBS on ice. Under a dissecting microscope, remove the remaining fat, and carefully remove the connective tissue capsule from the lacrimal gland. Place a sterile 35-millimeter Petri dish on ice.

Add 800 to 1000 microliters of cold PBS to the center of the dish and transfer one lacrimal gland into the PBS. Ensure the tissue remains submerged in PBS. Using two sterile scalpels, finely mince the lacrimal gland into small fragments until it reaches a soft paste-like consistency.

Collect the tissue fragments to one side of the dish. Aspirate the residual PBS and wash the fragments with one milliliter of fresh cold PBS. Transfer the washed tissue fragments into a 12-well plate containing two milliliters per well of pre-warmed digestion medium.

Place the plate in a shaking water bath and incubate at 37 degrees Celsius and 60 revolutions per minute for 30 minutes. Using a wide-bore one-milliliter pipette tip, gently triturate the tissue 15 times. Return the plate to the shaking water bath and incubate for an additional 30 minutes.

Next, using a regular one-milliliter pipette tip, gently triturate the sample 10 times and incubate again for 30 minutes. Then gently triturate the sample 10 times and transfer the cell suspension into two-milliliter dolphin micro centrifuge tubes. Add one milliliter of cold blocking medium one, or BM1, to each well of the plate to collect the remaining digestion medium containing residual cells and place the plate on ice until the next step.

Centrifuge the tubes containing the cell suspension at 300 G for five minutes at four degrees Celsius. Discard the supernatant from each tube. Transfer the BM1 from each well of the plate containing residual cells into the corresponding tube.

Next, add 600 microliters of cold BM1 to each well to rinse the remaining cells and transfer the wash into the corresponding tube to combine with the cell suspension. Gently resuspend the cell suspension. Centrifuge the tubes again at 300 G for five minutes at four degrees Celsius and discard the supernatant.

Add one milliliter of Accutase to the cell pellets and gently resuspend the cells. Incubate the tube at 37 degrees Celsius and 60 revolutions per minute for five to 10 minutes. Using a one-milliliter pipette tip, gently triturate the sample 10 times.

Then add one milliliter of cold BM2 to the tube to terminate the enzymatic reaction by diluting the Accutase. Centrifuge the tube at 300 G for five minutes at four degrees Celsius and discard the supernatant. Add medium containing DNase 1 at a concentration of 200 units per milliliter to the tube to resuspend the cells and fill the tube to the top.

Incubate the tube for 10 minutes at room temperature, and gently resuspend once after five minutes. Meanwhile, place a 70-micrometer cell strainer over a 15-milliliter conical tube, and pre-wet the strainer with one milliliter of BM3. After incubation, transfer the cell suspension onto the strainer.

Add one milliliter of BM3 into the original collection tube to rinse it and transfer the rinse onto the same strainer. Wash the strainer with an additional five milliliters of BM3. Finally, determine cell concentration and viability of the filtered cell suspension using trypan blue stain and an automated cell counter.

Proceed with downstream applications, such as single-cell RNA sequencing for gene expression profiling, or flow cytometry-based cell sorting of fluorescently-labeled cell populations. UMAP analysis of single-cell RNA sequencing data identified well-represented epithelial and stromal populations in the lacrimal gland, including myoepithelial cells and fibroblasts, as well as diverse immune cell populations. Aged lacrimal glands contained abundant plasma and activated B cells expressing IGHM and IGHD.

Sequential flow cytometry gating enabled clear separation of EpCAM-positive, GFP-positive myoepithelial cells from EpCAM-negative, GFP-positive mural cells. GFP-positive fibroblasts expressing PDGFR alpha were EpCAM-negative, allowing discrimination from epithelial cells. In PDGFR alpha, EGFP mice, all GFP-positive cells also expressed collagen one, confirming fibroblast identity.

Flow cytometry analysis showed that 99.6%of GFP-positive cells corresponded to PDGFR alpha immunostained cells. This protocol is designed to isolate viable single cells from lacrimal glands for molecular characterization of epithelial, stromal, and immune populations. The main challenge is minimizing mechanical and enzymatic stress during disassociation of fragile aged tissue, while also maintaining high cell viability for single-cell RNA sequencing.

Following this procedure, isolated cells can be used for single-cell RNA sequencing, bulk RNA sequencing, and molecular profiling of specific cell populations. Sorted cells can also be cultured for downstream functional assays.