We thank Dr. Ivo Kalajzic for providing us with the SMACreErt2 mouse strain, Takeshi Umazume for mouse tailing and genotyping, Mark Shelley for acquiring professional pictures for Figure 2. We also thank Scripps Council of Scientific Editors and Mark Shelley for Scientific English editing. We are grateful to The Scripps Research Institute Flow Cytometry core for assistance with cell sorting and to Dr. Robin Willenbring for multiple discussions/advice on FACS data analysis.

This work was supported by the National Institutes of Health, National Eye Institute Grants 5 R01 EY026202 and 1 R01 EY028983 to H.P.M.

All animal work was conducted according to the National Institute of Health (NIH) guidelines and was approved by Institutional Animal Care and Use Committee of the Scripps Research Institute. All efforts were made to minimize the number of mice and their suffering. All experimental animals received a standard diet with free access to tap water.
NOTE: The main steps for MEC and pericyte isolation are outlined schematically in Figure 1A-F. All reagents and equipment used for this procedure are described in Table 1.
1. Mice and Labelling the SMA cells
<ol>
	<li>Use adult (2-4 months old) tamoxifen-inducible, &#945;SMA driven reporter mice SMACreErt2/+:Rosa26-TdTomatofl/fl. 
	NOTE: The SMACreErt2 strain was kindly provided by Dr. Ivo Kalajzic13. Rosa26-TdTomatofl/fl (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, also known as Ai9) strain (# 007909) were purchased from Jackson Laboratory (Sacramento, CA). SMA+ cells were labeled by intraperitoneal tamoxifen (TM) administration.</li>
	<li>Preparation of tamoxifen solution
	<ol>
		<li>Prepare filtered corn oil. Use 0.22 &#181;m vacuum filter since corn oil is viscous.</li>
		<li>Transfer 1 g of TM powder from the bottle into a 50 mL tube. Add 1 mL of ethanol to the bottle, cap and shake it to rinse then add to a 50 mL tube. Repeat once more with another 1 mL of ethanol.</li>
		<li>Add filtered corn oil to make 50 mL of a 20 mg/mL TM solution. Vortex the tube, wrap it in foil, and put it in a shaking water bath or shaking incubator at 45 &#176;C.</li>
		<li>It may take about 12-24 h to dissolve the TM. From time to time, remove the tube and check for any remaining crystals. Once the TM is completely dissolved, aliquot and store at -80 &#176;C. A thawed aliquot can be reused.</li>
	</ol>
	</li>
	<li>To label SMA+ cells, inject mice intraperitoneally (IP) with TM on two sequential days.
	<ol>
		<li>Inject 3-4 weeks old SMACreErt2/+:Rosa26-TdTomatofl/f any gender mice with TM at 100 &#181;L/20 g (or 2 mg/20 g) body weight (Figure 1A). Mice are ready to be used for cell isolation in 2-3 days after the last TM injection. If needed, injected mice can be sacrificed at longer periods of time after TM injection. 
		NOTE: As controls for proper compensation during FACS, one wild type (C57Bl/6) mouse and one SMACreErt2/+:Rosa26-TdTomatofl/fl mouse not injected with TM (with &#8220;unstained&#8221; MECs) of the same age would be required. Use the same calculations provided for 2 SMACreErt2/+:Rosa26-TdTomatofl/fl mice. Not injected SMACreErt2/+:Rosa26-TdTomatofl/fl will allow evaluation of the DSRed background. The C57Bl/6 mouse will serve as a negative control of unstained cells.</li>
	</ol>
	</li>
</ol>
2. Solutions and Buffers
NOTE: The LG is an epithelial origin gland that contains an extracellular matrix that makes dissociation of cells difficult. Therefore, using a special combination of enzymes and a multistep digestion process described below is recommended.
<ol>
	<li>Dispase type II stock solution (25x)
	<ol>
		<li>Dissolve 120 mg of dispase type II powder in 2 mL of 50 mM HEPES/150 mM NaCl to prepare a 25x stock solution (final concentration of dispase should be 30 Units/mL). Units per milligram may vary depending on the number of mice and concentration of the dispase should be adjusted accordingly.</li>
		<li>Prepare 200 &#181;L aliquots and store them at -70 &#176;C for up to 6 months or 4 &#176;C for several days. Do not freeze/thaw the aliquot of dispase more than once to prevent enzyme degradation.</li>
	</ol>
	</li>
	<li>DNase type I stock solution
	<ol>
		<li>Dissolve 5 mg of DNase type I powder in a 5 mL solution of 50% glycerol, 20 mM Tris buffer (pH 7.5), and 1 mM MgCl2 (stock concentration should be approximately 2000 Units/mL). Units per milligram may vary depending on number of mice and thus concentration of DNase should be adjusted accordingly.</li>
		<li>Filter the stock solution using a 0.22 &#181;m filter and a 10 mL syringe.</li>
		<li>Prepare 200 &#181;L aliquots and store them at -70 &#176;C for up to 6 months or 4 &#176;C for several days. Do not freeze/thaw more than once to prevent enzyme degradation.</li>
	</ol>
	</li>
	<li>Digestion medium
	<ol>
		<li>To 10 mL of DMEM low glucose without glutamine, add 100 &#181;L of cell culture supplement (e.g., Glutamax, see Table of Materials) for a dilution of 1:100.</li>
		<li>To 2 mL of DMEM low glucose with cell culture supplement, add 6 mg of Collagenase type I and mix thoroughly by pipetting (enzyme on wet ice), 160 &#181;L of dispase stock solution (2.4 U/mL final concentration), 16 &#181;L of DNase type I stock solution (8 U/mL final concentration), and 12 &#181;L of 1 M CaCl2 (6 mM final concentration). 
		NOTE: Calcium is required to increase enzymatic activity14,15. All calculations are provided for isolation of cells from four lacrimal glands from two adult mice. The volume of digestion medium may vary depending on the amount of tissue and number of replicates. Do not use more than 4 lacrimal glands from 2-4 months old mice per 2 mL medium.</li>
	</ol>
	</li>
	<li>Blocking medium I
	<ol>
		<li>To 25 mL of DMEM/F-12, add FBS (15% final concentration), 250 &#181;L of cell culture supplement (see Table of Materials) for a dilution of 1:100, and 50 &#181;L of 0.5 M EDTA pH 8.0 (1 mM final concentration). 
		NOTE: Of the different types of medium that were compared for this protocol DMEM/F-12 gave the best results. This medium has also been used by other researchers to isolate/culture epithelial cells16,17.</li>
	</ol>
	</li>
	<li>Blocking medium II
	<ol>
		<li>To 25 mL of PBS, add 50 &#181;L of 0.5 M EDTA pH 8.0 (1 mM final concentration).</li>
	</ol>
	</li>
	<li>Recovery medium
	<ol>
		<li>To 2 mL of HBSS supplemented with 5 mM MgCl2, add 100 &#181;L of DNase type I stock solution to 100 U per 2 mL final concentration. Relatively high concentrations of DNase-type I is required to reduce aggregation of epithelial cells.</li>
	</ol>
	</li>
	<li>Fluorescence activated cell sorting (FACS) buffer
	<ol>
		<li>To 486.5 mL of PBS, add 12.5 mL of serum (2.5% final concentration) and 1 mL of 0.5 M EDTA pH 8.0 (1 mM final concentration). 
		NOTE: The buffer can be stored at 4 &#176;C for a maximum of 6 weeks.</li>
	</ol>
	</li>
</ol>
3. Adult Mouse Lacrimal Gland Harvesting and Microdissection
<ol>
	<li>Anesthetize the mouse by isoflurane inhalation (adjust the isoflurane flow rate or concentration to 5% or greater) and sacrifice by cervical dislocation. Perform anesthesia and euthanasia according to institutional IACUCs recommendations.</li>
	<li>Using fine forceps and scissors, remove skin between the eye and ear (Figure 2A).</li>
	<li>To dissect a LG, gently pull LG using tweezers and at the same time scratch the connective tissue around the LG using the sharp tip of small scissors to free it (Figure 2B).</li>
	<li>Avoid cutting with scissors, as the LG and parotid salivary glands are located very close to each other and must be separated prior to dissection. When LG and parotid glands are separated, cut the LG out using scissors. Place glands into a 35 mm dish with 2 mL of cold PBS (keep on ice) (Figure 1B).</li>
	<li>As the LG is covered by a connective tissue capsule/envelope, trim any surrounding fat and connective tissue under a dissecting microscope and remove the LG capsule with two forceps.
	<ol>
		<li>Repeat this step for all glands.</li>
	</ol>
	</li>
	<li>Check a small piece of tissue under fluorescent microscope to ensure cell labeling (Figure 1C).</li>
</ol>
4. Preparation of LG Single-cell Suspension
<ol>
	<li>Transfer all LGs into a 35 mm dish with 0.5 mL of room temperature (RT) digestion media and mince LGs using small scissors into very small pieces (approximately 0.2-1 &#181;m2). Normally, it takes about 3 min to mince 4 LGs (Figure 2C).</li>
	<li>Transfer minced tissue into a 2 mL round bottom tube using a wide-bore pipette filter tip. Use a normal sized pipette tip with the tip cut off (Figure 2D).</li>
	<li>Add up to 2 mL digestion medium and mix by inverting the tube.</li>
	<li>Place tube in a shaking incubator (or shaking water bath), at 37 &#176;C, 100-120 rpm for 90 min.</li>
	<li>Every 30 min slowly pipette gland pieces 20-30 times using a 1,000 &#181;L filter tip with the decreasing bore size (Figure 2D). After incubation/trituration, take a 10 &#181;L aliquot and inspect under a microscope for clusters. If clusters persist, continue digestion.</li>
	<li>After 90 min, pass the sample 2-3 times through an insulin syringe needle (31G) to further release cells into suspension. 
	NOTE: No visible lacrimal gland pieces should remain in the solution once digestion is completed (Figure 1D).</li>
	<li>Transfer cell suspension to a 15 mL tube and add blocking media type I to a total of 5 mL. Invert the tube 2-3 times to mix.</li>
	<li>Pass the cell suspension through a 70 &#956;m cell strainer placed on a 50 mL tube. Wash the strainer with 1 mL of blocking media type I. Repeat step 4.8 again.</li>
	<li>Centrifuge samples at 0.4&#160;x g for 5 min at RT.</li>
	<li>Aspirate the supernatant. Re-suspend cells in 2 mL of blocking medium type II using a 1 mL pipette tip and transfer the cell suspension into a 2 mL microcentrifuge tube.</li>
	<li>Centrifuge the cells at 0.4 x g (24 x 1.5/2.0 mL rotor; see Table of Materials) for 3 min at RT.</li>
	<li>Aspirate supernatant and re-suspend cells in 1 mL of cell detachment solution (see Table of Materials). 
	NOTE: Here, the cell detachment solution is Accutase, a marine-origin enzyme with proteolytic and collagenolytic activity that detaches/dissociates cells for analysis of cell surface markers.</li>
	<li>Incubate cells at 37 &#176;C, at 100-120 rpm for 2-3 min. Over-digestion with cell detachment solution may damage cellular membranes.</li>
	<li>Transfer cell suspension into a 50 mL tube and add up 10 mL of blocking medium type I. Centrifuge tube at 0.4 x g (24 x 1.5/2.0 mL rotor; see Table of Materials) for 5 min.</li>
	<li>Discard supernatant and re-suspend cells in 6 mL of recovery media and incubate cells for 30 min at RT.</li>
	<li>Check 10 &#181;L of cell suspension under the microscope to ensure complete cell dissociation (Figure 3).</li>
	<li>Count cells using a cell counter and Trypan blue. Normally, we expect 4 x 105-6 x 106 cells from four LGs (one sample).</li>
	<li>Centrifuge cells at 0.4 x g (24 x 1.5/2.0 mL rotor; see Table of Materials) for 3 min at RT and proceed to antibody staining.</li>
</ol>
5. Antibody Staining
<ol>
	<li>Add up to 5 x105 cells to a 2 ml tube containing 400 &#181;L of FACS buffer. Add 5 &#181;L of Brilliant Violet 421 anti-mouse CD326 (EpCAM) and 0.5 &#181;L of Ghost Red 780 (Viability Dye).</li>
	<li>In parallel, prepare controls to adjust FACS compensation: 
	Negative control-1 (cells from wild type mouse) 
	Background control unstained cells from SMACreErt2/+:Rosa26-TdTomatofl/fl 
	Cy7-780 stained cells (cells from the wild type mouse stained with the Ghost Red 780 Viability Dye) 
	EpCAM-Brilliant Violet 421 (cells from wild type mouse stained with the EpCAM-Brilliant Violet 421 antibody). 
	NOTE: For each control sample use a minimum of 1 x 105 cells per 400 &#181;L of FACS buffer.</li>
	<li>After adding each reagent mix cells thoroughly by pipetting.</li>
	<li>Wrap tube(s) with foil and rotate tubes for 45 min at 4 &#176;C.</li>
	<li>Centrifuge samples at 0.4 x g (24 x 1.5/2.0 mL rotor; see Table of Materials) for 3 min at 4&#176;C.</li>
	<li>Re-suspend cells in 1 mL of FACS buffer. It is important to wash cells to decrease the background during compensation.</li>
</ol>
6. Fluorescence Activated Cell Sorting
<ol>
	<li>Transfer cell suspension into 5 mL FACS tubes and proceed with FACS analysis. Keep cells on ice.</li>
	<li>Adjust compensation using single color controls.</li>
	<li>Sort cells at 20 psi through a 100 &#956;m nozzle using appropriate flow cytometer (see Table of Materials). Gating strategy18 is shown in Figure 1E and Figure 4.</li>
	<li>Collect sorted cells into medium, RNA-later, FACS or lysis buffers depending on downstream procedures (Figure 1E,F).</li>
</ol>

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The authors declare no competing financial interests and no conflicts of any other interests.

This manuscript described a protocol of MEC and pericyte isolation from LG and SMG. This procedure was based on genetic labeling of SMA, the only reliable biomarker of MECs and pericytes.
The urgency to develop this protocol was motivated by the almost total absence of literature highlighting the isolation of MECs from murine LGs and SMGs. Although genetic labeling was previously used, using SMA-GFP mice to isolate SMA+ cells from young three-week-old LGs12, it did not ...

Myoepithelial cells (MECs) are present in many exocrine glands including lacrimal, salivary, harderian, sweat, prostate, and mammary. MECs are a unique cell type that combines an epithelial and a smooth muscle phenotype. MECs express &#945;-smooth muscle actin (SMA) and have a contractile function1,2. In addition to MECs, the lacrimal gland (LG) and the submandibular gland (SMG) contains SMA+ vascular cells called pericytes, which are cells of endodermal origin that envelop the surface of vascular tubes3. Although MECs and pericytes express many markers, SMA is the only marker that is not expressed in other LG and SMG cells1,3.
Within the last 40 years, several laboratories reported assays for dissociation of different exocrine gland tissues, in which non-enzymatic and enzymatic approaches were applied. In one of the first reports published in 1980, Fritz and coauthors described a protocol to isolate feline parotid acini using sequential digestion in a collagenase/trypsin solution4. In 1989, Hann and coauthors adjusted this protocol for acini isolation from rat LGs using a mixture of collagenase, hyaluronidase and DNase5. In 1990, Cripps and colleagues published the method of non-enzymatic dissociation of lacrimal gland acini6. Later, in 1998, Zoukhri and coauthors returned to an enzymatic dissociation protocol for following up Ca2+-imaging on LG and SMG isolated acini7. Within the last decade, researchers have turned their focus on isolation of stem/progenitor cells from exocrine glands. Pringle and coauthors described a protocol in 2011 for isolation of mouse SMG stem cells8. This method was based on isolation of stem cell-containing salispheres, which were maintained in culture. The authors claimed that proliferating cells expressing stem cell-associated markers could be isolated from these salispheres8. Shatos and coauthors published the protocol for progenitor cell isolation from uninjured adult rat LGs using enzymatic digestion and collecting &#8220;liberated&#8221; cells9. Later, in 2015, Ackermann and coauthors adjusted this procedure to isolate presumptive &#34;murine lacrimal gland stem cells&#34; (&#34;mLGSCs&#34;) that could be propagated as a mono-layer culture over multiple passages10. However, none of the before mentioned procedures allowed for distinguishing cellular subtypes and individual populations of isolated epithelial cells. In 2016, Gromova and coauthors published a procedure for isolation of LG stem/progenitor cells from adult murine LGs using FACS11. However, this protocol was not intended to isolate MECs.
Recently, we have shown that we are able to isolate SMA+ cells from 3 week-old SMA-GFP mice12. However, at this time we have not separated different populations of SMA+ cells. Here we established a new procedure for the direct isolation of differentiated MECs and pericytes from adult LGs and SMGs.

<table><tbody><tr><td>Biosafety Cabinet</td><td>SterilCard Baker</td><td>19669.1</td><td>Class II type A/B3</td></tr><tr><td>10 mL Disposable serological pipets</td><td>VWR</td><td>89130-910</td><td>Manufactured from polystyrene and are supplied sterile and plugged</td></tr><tr><td>10 mL Disposable serological pipets</td><td>VWR</td><td>89130-908</td><td>Manufactured from polystyrene and are supplied sterile and plugged</td></tr><tr><td>15 mL High-clarity polypropylene conical tubes</td><td>Falcon</td><td>352196</td><td></td></tr><tr><td>25 mL Disposable serological pipets</td><td>VWR</td><td>89130-900</td><td>Manufactured from polystyrene and are supplied sterile and plugged</td></tr><tr><td>5 mL FACS round-bottom tubes</td><td>Fisher Scientific, Falcon</td><td>14-959-11A</td><td></td></tr><tr><td>50 mL High-clarity polypropylene conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Antibiotic-antimycotic</td><td>Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>Appropriate filter and non-filter tips</td><td>Any available</td><td>Any available</td><td></td></tr><tr><td>BD Insulin Syringes</td><td>Becton Dickinson</td><td>328468</td><td>with BD Ultra-Fine needle &amp;frac12; mL 8 mm 31 G</td></tr><tr><td>BD Syringes 10 mL</td><td>Becton Dickinson</td><td>309604</td><td>Sterile</td></tr><tr><td>Brilliant Violet 421 anti mouse CD326 (EpCAM)</td><td>Biolegend</td><td>118225</td><td>Monoclonal Antibody (G8.8)</td></tr><tr><td>CaCl&lt;sub&gt;2 &lt;/sub&gt;1M solution</td><td>BioVision</td><td>B1010</td><td>sterile</td></tr><tr><td>Cell culture dishes 35 mm</td><td>Corning</td><td>430165</td><td>Non-pyrogenic, sterile</td></tr><tr><td>Collagenase Type I</td><td>Wortington</td><td>LS004194</td><td></td></tr><tr><td>Corn oil</td><td>Any avaliable</td><td>Any avaliable</td><td>From grocery store</td></tr><tr><td>Corning cell strainer size 70 &amp;mu;m</td><td>Sigma-Aldrich</td><td>CLS431751-50EA</td><td></td></tr><tr><td>Digital Stirrer PC-410D</td><td>Corning</td><td>Item# UX-84302-50</td><td></td></tr><tr><td>Dispase II</td><td>Sigma-Aldrich</td><td>D4693-1G</td><td></td></tr><tr><td>Dissecting scissors, curved blunt</td><td>McKesson Argent</td><td>487350</td><td>Metzenbaum 5-1/2 Inch surgical grade stainless steel non-sterile finger ring handle</td></tr><tr><td>DNase I</td><td>Akron Biotech, catalog number</td><td>AK37778-0050</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium &amp;ndash; low glucose (DMEM)</td><td>Sigma-Aldrich</td><td>D5546-500ML</td><td>with 1,000 mg/L glucose and sodium bicarbonate, without L-glutamine</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium/F12 (DMEM/F12)</td><td>Millipore</td><td>DF-042-B</td><td>without HEPES, L-glutamine</td></tr><tr><td>Easypet 3 pipette controller</td><td>Eppendorf</td><td>4430000018</td><td>with 2 membrane filters 0.45 &amp;micro;m, 0.1 &amp;ndash; 100 mL</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023-500ML</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>Fisher Vortex Genie 2</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr><tr><td>FlowJo version 10</td><td>Any available</td><td>Any available</td><td></td></tr><tr><td>Fluorescence binocular microscope Axioplan2</td><td>Carl Zeiss</td><td>ID# 094207</td><td></td></tr><tr><td>Ghost Red 780 Viability Dye</td><td>Tonbo Biosciences</td><td>13-0865-T100</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>ThermoFisher Scientific, Gibco</td><td>35050061</td><td></td></tr><tr><td>Glycerol 99%</td><td>Sigma-Aldrich</td><td>G-5516</td><td></td></tr><tr><td>Hand tally counter</td><td>Heathrow Scientific</td><td>HEA6594</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Sigma Millipore</td><td>H6648-500ML</td><td>Modified, with sodium bicarbonate, without calcium chloride, magnesium sulphate, phenol red.</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>ThermoFisher Scientific</td><td>14025092</td><td>With calcium, magnesium, no phenol red.</td></tr><tr><td>Hausser Bright-Line Phase Hemocytometer</td><td>Fisher Scientific</td><td>02-671-51B</td><td>02-671-51B</td></tr><tr><td>HEPES 1 M solution</td><td>ThermoFisher Scientific, Gibco</td><td>15630-080</td><td>Dilute 1/10 in ddH20</td></tr><tr><td>HyClone Fetal Bovine Serum (FBS)</td><td>Fisher Scientific</td><td>SH3007002E</td><td></td></tr><tr><td>Hydrochloric Acid (HCl), 5 N Volumetric Solution</td><td>JT Baker</td><td>5618-03</td><td>To adjust Tris buffer pH</td></tr><tr><td>Innova 4230 Refrigerated Benchtop Incubator</td><td>New Brunswick Scientific</td><td>SKU#:</td><td>Shaker; 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; in air</td></tr><tr><td>Iris scissors</td><td>Aurora Surgical</td><td>AS12-021</td><td>Pointed tips, delicate, curved, 9 cm, ring handle</td></tr><tr><td>Isoflurane Inhalation Anesthetic</td><td>Southern Anesthesia Surgical (SAS)</td><td>PIR001325-EA</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;1 M solution</td><td>Sigma-Aldrich</td><td>63069-100ML</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 mL</td><td>ThermoFisher Scientific</td><td>3451</td><td>Clear, graduated, sterile</td></tr><tr><td>Microsoft Power Point</td><td>Any available</td><td>Any available</td><td></td></tr><tr><td>NaCl powder</td><td>Sigma-Aldrich</td><td>S-3014</td><td></td></tr><tr><td>Nalgene 25 mm Syringe Filters</td><td>Fisher Scientific</td><td>724-2020</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>pH 510 series Benchtop Meter</td><td>Oakton</td><td>SKU: BZA630092</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>ThermoFisher Scientific</td><td>10010023</td><td>pH 7.4</td></tr><tr><td>Pure Ethanol 200 Proof</td><td>Pharmco-Aaper</td><td>111000200</td><td></td></tr><tr><td>Red blood cell lysis buffer 10x</td><td>BioVision</td><td>5831-100</td><td></td></tr><tr><td>Roto-torque Heavy Duty Rotator</td><td>Cole Parmer</td><td>MPN: 7637-01</td><td></td></tr><tr><td>Safe-lock round bottom Eppendorf tubes 2 mL</td><td>Eppendorf Biopur</td><td>22600044</td><td>PCR inhibitor, pyrogen and RNAse-free</td></tr><tr><td>Scissors</td><td>Office Depot</td><td>375667</td><td></td></tr><tr><td>Sorting flow cytometer MoFlo Astrios EQ</td><td>Beckman Coulter</td><td>B25982</td><td>With Summit 6.3 software</td></tr><tr><td>Sorvall Legend Micro 17R Microcentrifuge</td><td>Thermo Scientific</td><td>75002441</td><td>All centrifugation performed at RT</td></tr><tr><td>Sorvall RT7 Plus Benchtop Refrigerated Centrifuge</td><td>Thermo Scientific</td><td>ID# 21550</td><td>RTH-750 Rotor. All centrifugation performed at RT</td></tr><tr><td>Stemi SV6 stereo dissecting microscope</td><td>Carl Zeiss</td><td>455054SV6</td><td>With transmitted light base</td></tr><tr><td>Tamoxifen</td><td>Millipore Sigma</td><td>T5648-1G</td><td></td></tr><tr><td>Trizma base powder</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Trypan blue solution</td><td>Millipore Sigma</td><td>T8154</td><td></td></tr><tr><td>Two Dumont tweezers #5</td><td>World Precision Instruments</td><td>500342</td><td>11 cm, Straight, 0.1 mm x 0.06 mm tips</td></tr><tr><td>Upright microscope</td><td>Any available</td><td>Any available</td><td>With transmitted light base</td></tr><tr><td>Vacuum filtration systems, standard line</td><td>VWR</td><td>10040-436</td><td></td></tr><tr><td>Variable volume micropipettes</td><td>Any available</td><td>Any available</td><td></td></tr></tbody></table>

isolation of myoepithelial cells from adult murine lacrimal and submandibular glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

Mouse model to isolate SMA+ MECs and pericytes 
The established protocol allows for the isolation of two pure populations: MECs and pericytes from LGs and SMGs (see Table 1). These two types of cells have a different size and appearance. Microvascular pericytes, develop around the walls of capillaries (Figure 5A) and have a squared shape (Figure 5B), while MECs surround the LG secretory acin...

Watch this Scientific Journal Video about Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands at JoVE.com

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

isolation-myoepithelial-cells-from-adult-murine-lacrimal

Research

JoVE Journal

Biology

8.0K Views.  The Scripps Research Institute. The lacrimal gland (LG) has two cell types expressing α-smooth muscle actin (αSMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Video: Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Immunology and Infection

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Developmental Biology

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue.