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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#189; mL 8 mm 31G</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>CaCl2 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 &#956;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&#8217;s Modified Eagle&#8217;s Medium &#8211; low glucose (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5546-500ML</td>
			<td>with 1000mg/L glucose and sodium bicarbonate, without L-glutamine</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;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 &#181;m, 0.1 &#8211; 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&#39;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&#39;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 1M 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), 5N 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 &#176;C, 5% CO2 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>MgCl2 1M 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 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

7.9K 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 Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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 ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...