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

Summary

This protocol describes an optimized method for dissociating aged mouse lacrimal glands to obtain viable single cells for single-cell RNA sequencing. Sequential enzymatic digestion and gentle handling preserve cell integrity, yielding high-quality suspensions suitable for cell-type-specific isolation and transcriptomic analysis of aging-associated changes in lacrimal gland populations.

Abstract

Aging of the lacrimal gland (LG) is associated with reduced tear secretion and chronic inflammation, contributing to dry eye disease. Age-related LG changes include immune cell infiltration, cellular damage, lipid accumulation in acinar cells, extracellular matrix deposition, and ductal dilation. Single-cell RNA sequencing (scRNA-seq) provides a powerful approach to profile cellular heterogeneity and transcriptional changes during aging; however, generating viable single-cell suspensions from aged LG tissue is technically challenging due to increased fibrosis, cellular fragility, and lipid accumulation. This study presents an optimized, reproducible protocol for the efficient dissociation of aged mouse LGs and the isolation of multiple cell types, suitable for downstream scRNA-seq analysis. Importantly, this protocol is also applicable to young lacrimal glands, conjunctiva, and other exocrine tissues. The workflow employs a stepwise enzymatic dissociation strategy using Collagenase IV, Dispase II, and DNase I for initial tissue digestion, followed by Accutase to dissociate remaining cell clusters and a final DNase I treatment to obtain a clean single-cell suspension. To minimize cell damage, all plasticware is pre-coated with PBS containing bovine serum albumin, and tissue processing is performed under controlled temperature and agitation conditions. Red blood cell lysis and sequential filtration through several strainers further enhance cell purity. Cell type-specific isolation is achieved using reporter mouse lines combined with fluorescence-activated cell sorting (FACS). Using Acta2^GFP and Pdgfra^EGFP mice, we successfully isolated myoepithelial cells and fibroblasts, respectively, following immunostaining with an EpCAM antibody. FACS enabled the separation of the EpCAM⁺/αSMA⁺ myoepithelial population, while EGFP⁺ cells from Pdgfra^EGFPmice represented the fibroblast population. This protocol yields high-quality, viable single-cell suspensions from aged LGs and can be readily adapted to other fibrotic or aging exocrine tissues requiring gentle dissociation and targeted cell isolation.

Introduction

The lacrimal gland (LG) is an exocrine tubuloacinar gland responsible for secreting the aqueous layer of the tear film. Throughout life, the corneal surface and the LG are exposed to bacterial and viral infections, which may lead to systemic disruption of epithelial cells, induction of chronic inflammatory responses, immune cell infiltration into the LG, and a lack of regeneration. Aging is accompanied by an increased incidence of chronic inflammatory diseases¹. Among these, dry eye disease (DED) is one of the ten most common causes of visual impairment worldwide and is a major contributor to decreased independence, mobility, and daily functioning in the elderly population². A primary cause of DED is LG dysfunction³.

With advancing age, the mouse and human LG undergo pronounced structural and functional alterations, including acinar cell atrophy, ductal dilation, immune cell infiltration, fibrosis, and lipid deposition^4,5,6,7. These degenerative changes are also accompanied by chronic inflammation of the LG and tissue fibrosis^3,5,8,9. Despite the clear association between aging and LG pathology, the molecular and cellular mechanisms driving these changes remain poorly understood.

Single-cell RNA sequencing enables comprehensive profiling of cell populations and their transcriptional changes during aging¹⁰. This approach allows unbiased identification of distinct cell types and states within the lacrimal gland, revealing how aging alters gene expression profiles, immune cell infiltration, and epithelial-mesenchymal interactions.

At present, numerous tissue dissociation methods are available, including mechanical dissociation methods¹¹ and protocols involving warm enzymatic digestion at 37 °C with enzymes such as Collagenase, Dispase, or Trypsin¹². Various dissociation strategies have been described for a wide range of tissues, including liver, lung, kidney, heart, and multiple tumor types^13,14,15. However, preparing viable single-cell suspensions from aged lacrimal gland tissue remains technically challenging because of increased fibrosis and cellular fragility. Also, as we have reported previously, recovery of fragile acinar cells can be challenging, as dissociation-associated changes in membrane permeability may increase ambient RNA release from these large, secretory cells, contributing to background signal in downstream single-cell datasets¹⁶. Although several studies have reported scRNA-seq atlases of aging lacrimal glands, the quality of these datasets has been limited by suboptimal dissociation methods, resulting in a lower yield of epithelial cells than of stromal populations. Here, we present a detailed, optimized protocol for the efficient dissociation of aged mouse LG and the isolation of distinct cell populations for downstream scRNA-seq and bulk RNA-seq analyses (Figure 1). This approach provides a powerful tool to study the molecular mechanisms underlying age-related lacrimal gland dysfunction.

The protocol enables the generation of high-quality, viable single-cell suspensions from aged lacrimal glands, providing a reliable foundation for comprehensive transcriptomic analyses. It allows detailed identification and molecular characterization of diverse cell populations, including myoepithelial cells, fibroblasts, and other epithelial and stromal cell types that undergo age-related changes. The optimized workflow preserves cell integrity and minimizes stress-induced transcriptional artifacts, ensuring accurate representation of in vivo cellular states. Moreover, the methodology is broadly applicable and can be adapted to other exocrine glands or fibrotic tissues, where gentle tissue dissociation and precise isolation of distinct cell populations are critical for downstream single-cell studies.

Protocol

All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at The Scripps Research Institute (protocol no. 08-0123; approved on August 29, 2023). The animal care at The Scripps Research Institute is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). Mice were maintained under a standard 12 h light/12 h dark cycle with food and water provided ad libitum. Pdgfra^EGFP and Acta2^GFP transgenic mouse lines were used for experiments. The detailed information on mouse lines is provided in the "Table of materials" section.

1. General considerations

1. Follow NIH Universal Precautions¹⁷ when handling biohazardous materials and animal tissues. Perform all animal work in accordance with approved IACUC protocols and designated Animal Biosafety Levels.
2. Follow standard laboratory safety practices by avoiding mouth pipetting, minimizing sharps use, using a biological safety cabinet for aerosol-generating procedures, wearing appropriate PPE, disposing of sharps in puncture-resistant containers, and properly labeling and decontaminating all biohazardous waste prior to disposal.
3. Routinely decontaminate work surfaces and equipment using appropriate disinfectants (e.g., 10% bleach).
4. Perform all centrifugation steps at 4 °C in a pre-cooled refrigerated centrifuge to preserve cell viability and minimize temperature-induced stress.
5. Optimize flow cytometer and cell sorter settings for the specific instrument configuration and adjust parameters for each antibody-fluorophore conjugate using appropriate control samples. Use the same cytometer and sorting instrument, with fixed, optimized settings, throughout an experimental series to minimize variability.
6. Prepare selected solutions up to one day in advance and store appropriately until use. Prepare all solutions under sterile conditions in a biosafety cabinet.
7. Keep Ca²⁺/Mg²⁺-containing solutions separate from EDTA-containing buffers to prevent chelation during enzymatic steps. All reagents needed for this procedure are shown in Table of Materials.

2. Plasticware

1. In sterile conditions, pre-coat dishes, tubes, and syringes with 1x PBS + 1% BSA for 1 h at room temperature (RT). Aspirate and keep everything sterile until use.
2. Pre-coat pipette tips. Aspirate and dispense 1x PBS + 1% BSA three times with each tip to coat the inner surface. Place the coated tips back into the sterile tip box and allow them to dry overnight in a sterile incubator. Use these pre-coated tips the next day during the cell isolation procedure.

3. Solutions and buffers

1. DNase I Stock (5 mg/mL)
   1. Prepare 150 mM NaCl from the 5M NaCl stock and sterile-filter the solution.
   2. Add 1 M CaCl₂ to the filtered 150 mM NaCl solution to obtain a final concentration of 5 mM CaCl₂.
   3. Resuspend 100 mg DNase I in 20 ml of 150 mM NaCl / 5 mM CaCl₂ buffer to obtain a 5 mg/mL DNase I stock solution.
   4. Prepare 500 µL aliquots and store them at −20 °C for up to 6 months. Avoid repeated freeze-thaw cycles to prevent loss of enzyme activity.
2. Digestion medium
   1. The day before the experiment, prepare the medium base mix by mixing DMEM/F12 with 0.5% BSA and filter it.
   2. Add 1M HEPES and 1M CaCl₂ to the base medium DMEM/F12 / 0.5% BSA to obtain final concentrations of 15 mM and 3 mM, respectively.
   3. Add all following enzymes right before use: Collagenase IV (1 mg/mL; 125 U/mL), Dispase II (2.5 mg/mL; 1.25 U/mL), and DNase I (20 U/mL; 0.05 mg/mL from the 5 mg/mL stock).
3. Blocking Medium 1 (BM1)
   1. Add 1M HEPES and 0.5M EDTA to 1x HBSS to obtain final concentrations of 15 mM and 3 mM, respectively.
4. Blocking Medium 2 (BM2)
   1. Add FBS and 1M HEPES to DMEM/F12 to obtain final concentrations of 10% and 15 mM, respectively.
5. Medium for DNase treatment
   1. The day before the experiment, prepare the base medium by mixing 1x HBSS with 0.5% BSA and filter it.
   2. Add 1M HEPES, 1M CaCl₂, and 1M MgCl₂ to the base medium 1x HBSS / 0.5% BSA to obtain final concentrations of 10 mM, 1.5 mM, and 2 mM, respectively.
   3. Right before use, add DNase I (200 U/mL; 0.5 mg/mL from the 5 mg/mL stock) into the prepared mix.
6. Blocking Medium 3 (BM3)
   1. Use the same filtered base medium 1x HBSS / 0.5% BSA and add 1M HEPES and 0.5M EDTA to obtain final concentrations of 10 mM and 1 mM, respectively.
7. Blocking Medium 4 (BM4)
   1. Use the same filtered base medium 1x HBSS / 0.5% BSA and add 1M HEPES to obtain final concentrations of 10 mM.
8. FACS Buffer
   1. The day before the experiment, prepare the base medium by mixing 1x DPBS with 0.5% BSA and filter it.
   2. Add 1M HEPES and 0.5M EDTA to 1x DPBS / 0.5% BSA to obtain final concentrations of 25 mM and 1 mM, respectively.
9. Medium for scRNAseq (MSC)
   1. The day before the experiment, prepare the medium by mixing 1x DPBS with 0.4% BSA and filter it.

4. Tissue harvesting and microdissection (~ 40 - 50 min)

1. Anesthetize the mouse with isoflurane and euthanize by cervical dislocation in accordance with institutional IACUC guidelines.
2. Use fine forceps and scissors to incise the skin between the eye and the ear and gently retract it to expose the lacrimal gland.
3. To isolate the gland, lift it slightly using a dull-tipped forceps and carefully separate the surrounding connective tissue by gently teasing it away in a circular motion. The boundaries of the lacrimal gland are clearly visible, allowing easy separation from adjacent tissues, including the parotid gland.
4. Transfer the lacrimal gland (each gland weighing approximately 10-25 mg, depending on mouse weight, sex, and age) to a 35 mm Petri dish containing 2 mL of cold 1x PBS on ice. Under a dissecting microscope, remove the remaining fat and carefully peel off the connective tissue capsule using two forceps.
5. Confirm cell labeling by examining a small tissue fragment under a fluorescence microscope and verifying the expected reporter or fluorescent signal before proceeding to enzymatic digestion.
6. Place a sterile 35 mm Petri dish on ice and add 800-1000 µL of cold 1x PBS (volume adjusted to gland size) to the center of the dish. Transfer one lacrimal gland into the 1x PBS.
7. Using two sterile scalpels, finely mince the gland into small fragments until it reaches a soft, paste-like consistency. Ensure that the tissue fragments remain submerged in PBS throughout the mincing process, but do not allow them to float freely across the dish.
8. Collect tissue fragments at one side of the dish, aspirate residual PBS, and wash fragments with 1 mL of fresh cold PBS.
9. Transfer washed tissue fragments into a 6-well plate containing 2 mL per well of pre-warmed digestion medium. Keep the plate on ice until all samples are processed.

5. First enzymatic digestion (~1.5 - 2 h)

1. Incubate the plate in a shaking water bath at 37 °C, 60 rpm for 30 min.
2. Gently triturate the tissue 15 times using a wide-bore 1 mL tip, then return the plate to the shaking water bath at 37 °C, 60 rpm for an additional 30 min.
3. Gently triturate the sample 10 times using a regular 1 mL tip, then return the plate to the shaking water bath at 37 °C, 60 rpm for an additional 30 min.
4. Gently triturate the sample 10 times and transfer the cell suspension into 2 mL Dolphin microcentrifuge tubes.
5. Add 1 mL of cold BM1 to each well of the plate to collect the remaining digestion medium containing residual cells. Place the plate on ice until the next step.
   NOTE: If large cell clusters or tissue fragments are still observed at this stage, extend each incubation step by an additional 10-20 min. Do not exceed the indicated number of trituration passes (maximum 10-15 gentle passes), as additional mechanical stress will compromise cell viability.

6. Termination of enzymatic activity and primary washes (~ 15 min)

1. Centrifuge the tubes containing the cell suspension at 300 × g for 5 min at 4 °C.
2. Discard the supernatant. Transfer the BM1 from each well of the initial plate (containing residual cells) into the corresponding tube and gently resuspend the cell pellets.
3. Rinse each well with an additional 600 µL of cold BM1 and combine the wash with the corresponding tube to collect all remaining cells.
4. Gently resuspend the cell suspension, centrifuge again at 300 × g for 5 min at 4 °C, and discard the supernatant.

7. Accutase and DNase treatment (~ 30 - 35 min)

1. Add 1 mL of Accutase to the cell pellets and gently resuspend. Incubate the tube for only 5-10 min at 37 °C and 60 rpm.
   NOTE: Place the tube on its side during incubation to prevent cells from settling and sticking to the bottom.
2. Gently triturate 10 times using a 1 mL pipette tip.
3. Add 1 mL of cold BM2 to terminate the enzymatic reaction by diluting the Accutase.
4. Centrifuge at 300 × g for 5 min at 4 °C and discard the supernatant.
5. Resuspend the cells in the medium for DNase treatment, filling the tube up to the top.
6. Incubate for 10 min at RT, gently resuspending once after 5 min.
7. Meanwhile, pre-wet a 70 µm cell strainer with 1 mL of BM3 placed over a 15 mL conical tube.
8. Transfer the cell suspension onto the strainer.
9. Rinse the original collection tube with 1 mL of BM3 and transfer the rinse onto the same strainer.
10. Wash the strainer with an additional 5 mL of BM3.

8. Cell counting and viability check

1. Take 10 µL of the filtered cell suspension in BM3 and mix with 10 µL of Trypan Blue. Determine cell concentration and viability using Trypan Blue exclusion and quantify by manual counting or an automated cell counter (e.g., Countess 3 Automated Cell Counter). Proceed further only if viability meets the required threshold for downstream applications (typically ≥70-80%).
   NOTE: Consider the total volume ~7-9 mL now in the 15 mL tube when calculating yield.
2. Proceed according to the downstream application. For scRNA-seq, continue to Section 9. For flow cytometry-based cell sorting, continue to Section 10.
   NOTE: The scRNA-seq workflow includes RBC lysis and magnetic dead-cell removal to reduce debris and ambient RNA and improve data quality. However, these steps may bias cell-type representation by preferentially affecting fragile or rare epithelial populations, including acinar cells. For flow cytometry-based sorting, these steps were omitted to maximize recovery and preserve cell-surface epitopes. Workflow selection should therefore be guided by experimental goals, prioritizing data quality for scRNA-seq or cell recovery and viability for sorting and downstream applications.

9. Preparation of cells for scRNA-seq (~ 1 - 1.5 h)

1. Prepare 1x RBC solution by reconstituting 10x solution with milliQ water.
2. Centrifuge the 15 mL tube containing the cell suspension at 400 × g for 5 min at 4 °C and discard the supernatant.
3. Resuspend the cell pellet in 5 mL 1x RBC buffer and incubate for 5 min at RT.
4. Centrifuge again at 300 × g for 5 min at 4 °C and discard the supernatant.
5. Resuspend the cell pellet in 2 mL cold BM4.
6. Take 10 µL of the filtered cell suspension in BM4 and mix with 10 µL of Trypan Blue to assess cell concentration and viability.
7. Pre-wet the MACS 30 µm strainer with 1 mL 1x HBSS and filter the cell suspension through the strainer into a 15 mL tube.
8. Rinse the previous collection tube with 2 mL of cold BM4 and pass the wash through the same strainer into the 15 mL tube.
9. Wash the strainer with an additional 10 mL cold BM4.
10. Centrifuge the 15 mL tube at 400 × g for 5 min at 4 °C and completely remove the supernatant.
11. Gently resuspend the pellet in 100 µL of MACS Microbeads and incubate for 15 min at RT.
12. Meanwhile, prepare 1x Binding Buffer (BB) from Dead Cell Removal Kit using sterile double-distilled water.
13. Wash a MACS MS column with 500 µL 1x BB to equilibrate.
14. Add 400 µL BB to the sample, resuspend the cells with Microbeads and apply the entire volume onto the equilibrated MS column. Collect the flow-through into a 15 mL tube.
15. Wash the column four times with 500 µL BB each, collecting all flow-through fractions.
16. Centrifuge the collected flow-through at 400 × g for 5 min at 4 °C and discard the supernatant.
17. Resuspend the cells in 400 µL of MSC buffer and transfer the suspension to a 1.5 mL tube.
18. Rinse the previous tube with 700 µL MSC buffer and pool the rinse with the sample.
19. Centrifuge at 400 × g for 5 min at 4 °C and discard the supernatant.
20. Resuspend the cell pellet in MSC buffer using a wide-bore tip up to a final volume of around 1.5 mL.
21. Centrifuge again at 400 × g for 5 min at 4 °C and discard the supernatant.
22. Leave ~100 µL of supernatant, gently resuspend the pellet, and count the cells again by mixing 6 µL cells with 6 µL Trypan Blue.
23. Adjust the final volume with MSC buffer to achieve 700-1200 cells/µL and recount cells to confirm cell concentration.
24. Ensure cell viability after RBC lysis and dead-cell removal is ≥ 70% (typically 70-95%) prior to loading for 10x Chromium processing (Chromium Single Cell 3' Solution V3).
25. Apply basic scRNA-seq quality control criteria: nUMI > 500, nGene > 200, and mitochondrial gene fraction <15%. Use a permissive mitochondrial threshold (15%) to preserve epithelial populations with higher metabolic activity. Include only cells meeting all QC criteria in downstream analyses.
    NOTE: These QC thresholds represent example values derived from our datasets (Supplementary Figure 1) and were reported in our previous publication¹⁸.

10. Preparation of cells for flow cytometry-based cell sorting (~ 1 - 1.5 h)

1. For flow cytometry-based isolation of MECs and fibroblasts, Pdgfra^EGFP and Acta2^GFP reporter mouse lines were used, respectively, to enable precise identification of each population.
2. Centrifuge the 15 mL tube with cell suspension at 400 × g for 5 min at 4 °C and discard supernatant.
3. Resuspend the cell pellet in cold FACS buffer to a final concentration of 5 x 10⁵ to 2 x 10⁶ cells per 100 µL.
4. Prepare the controls required for FACS compensation: negative control - unstained cells from a wild-type mouse, background fluorescence control - unstained cells from Acta2^GFP and Pdgfra^EGFP mice, single-stain control - cells from a wild-type mouse stained individually with each antibody used in the experiment.
   NOTE: For each control sample, use at least 1 × 10⁵ cells resuspended in 100 µL of FACS buffer.
5. Add FcBlock (1:100) and incubate for 5 min on ice.
6. Add CD326 (EpCAM)-APC monoclonal antibody (1:100) or CD140a (PDGFRα) monoclonal antibody (1:100). Incubate for 45 min on ice, protected from light.
7. Gently resuspend the cells every 15 min during antibody incubation.
8. Fill each tube to the top with cold FACS buffer and centrifuge at 300 × g for 5 min at 4 °C.
9. Prepare FxCycle working solution (1:1000) in FACS buffer.
10. Resuspend samples in 1 mL and controls in 500 µL of cold FACS buffer with FxCycle.
11. Transfer all samples to FACS tubes and keep them on ice, protected from light.
12. Proceed immediately to the Flow Cytometry Core for sorting. Collect sorted cells into FACS buffer, culture medium, lysis buffer, or RNAlater, depending on downstream applications.
    NOTE: Instrument settings (e.g., nozzle size and pressure) and event rates depend on the sorter configuration and target cell population; therefore, a preliminary pilot run is recommended to determine optimal parameters for each experiment.

Results

Dissociating aged exocrine glands is challenging due to lipid accumulation and extensive ECM deposition. Aged glands exhibit marked lymphocytic infiltration and pronounced ductal dilation with intraluminal material, reflecting the structural and inflammatory changes that complicate tissue dissociation. The lacrimal gland is an exocrine organ composed of three principal epithelial cell types: acinar, ductal, and myoepithelial cells. Acinar cells produce the aqueous and protein components of the tear film that maintain cor...

Discussion

Here, we optimized and evaluated a significantly improved protocol for generating single-cell suspensions from exocrine tissues, focusing on the lacrimal gland. Using this approach, we generated clean, high-quality single-cell suspensions from both old and young LGs, suitable for single-cell sequencing and other downstream applications.

Despite the availability of numerous tissue dissociation protocols, it was necessary for us to modify and optimize them to efficiently recover those cell popu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
BSA	Sigma-Aldrich	A4919
CaCl2 (1 M solution)	BioVision	B1010-100
Collagenase IV	Sigma-Aldrich	C5138
Dead Cell Removal Kit	Miltenyi biotec	130-090-101
Dead Cell Removal Kit	Miltenyi biotec	130-090-101
Dispase II	Roche	C756V28
DNase I	Sigma-Aldrich	DN25-100MG
DPBS	Gibco, Thermo Fisher Scientific	14190-144
Fetal Bovine Serum, ultra-low IgG	Gibco	# 16250078
Hank's Balanced Salt Solution (HBSS)	Corning (Thermo Fisher Scientific)	21-022-CV
HEPES (1 M solution)	Sigma-Aldrich	H0887
Isoflurane Solution	Covertus	# 11695067772
MACS® SmartStrainers (30 µm)	Miltenyi biotec	130-098-458
MACS® SmartStrainers (70 µm)	Miltenyi biotec	130-110-916
Medium/F12 (DMEM/F12)	Millipore	DF-042-B
MgCl2 1M	Sigma-Aldrich	M1028-100ML
MS Columns	Miltenyi biotec	130-042-201
NaCl (5M stock)	Invitrogen™	AM9759
Phosphate Buffered Saline (PBS) tablets	Sigma-Aldrich	P8920-500ML
Red Blood Cell Lysis Solution (10×)	Miltenyi biotec	130-094-183
StemPro Accutase	Gibco, Thermo Fisher Scientific	A1110501
Trypan Blue Solution, 0.4%	Gibco, Thermo Fisher Scientific	15250061
Ultra-pure EDTA (0.5M, pH 8.0)	Invitrogen	15575020
Antibodies
CD140a (PDGFRA) Monoclonal Antibody (APA5), APC-eFluor™ 780, Rat / IgG2a, kappa	eBioscience	47-1401-82; RRID:AB_2784706
FcBlock (CD16/CD32 Fc blocker)	BD Biosciences	553141; RRID:AB_394656
FxCycle™ Violet Stain	Thermo Fisher Scientific	F10347
Rat CD326 (EpCAM)-APC monoclonal antibody (G8.8), rat IgG2a, kappa	eBioscience	17-579182; RRID:AB_2716944
Mice
Acta2GFP	Kind gift from Dr. Ivo Kalajzic	PMID: 18571490
PdgfraEGFP  - B6.129S4-Pdgfratm11(EGFP)Sor/J	The Jackson laboratory	RRID:IMSR_JAX:007669
Equipment
Invitrogen™ Countess™ 3 Automated Cell Counter	Fisher Scientific	A49862; RRID:SCR_026963