The authors have no acknowledgments.

All vertebrate animal use protocols have been approved by the University of Minnesota Institutional Animal Care and Use Committee in accordance with recommended NIH and government guidelines.
1. Feeder Layer Initialization and Subculturing
<ol>
	<li>Thaw one cell vial containing frozen Swiss mouse 3T3 from the liquid nitrogen tank by placing the vial in a 37 &#176;C water bath for 1-2 min. The number of cells in the vial is approximately 1 x 106.</li>
	<li>Remove the 3T3 cell vial when the final sliver of ice has melted. Wipe the tube with a 70% alcohol swab. Then, transfer the vial to a biosafety cabinet.</li>
	<li>Transfer the cells slowly into a T-150 flask, and rinse the 3T3 vial with 1 mL of CGM medium. Slowly add 30 mL of pre-warmed 3T3 Complete Growth Medium (CGM) to the cells. Gently rock the flask to evenly spread the 3T3 fibroblast cells. Label the flask with cell line details, date, and passage number appropriately.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 and 95% humidity. 
	NOTE: According to the ATCC, the doubling time of Swiss mouse 3T3 is 18 h; when confluent (contact inhibited), the density is about 40,000 cells per cm2. We use a particular line of 3T3 within 10-15 passages after initialization. More rapid growth or the presence of spindle-shaped cells can occur at higher passages and is usually a sign that the 3T3 will not perform as well as feeder cells for adult mouse keratinocytes. If rapid growth occurs, it is better to initiate a new line from lower passage cells.</li>
	<li>Change media after 24 h to remove dead cells and remaining DMSO (cryopreservation agent), and twice weekly thereafter. Allow cells to proliferate around 80% confluence and use T-150 flasks for culture initiation. Use T-225 flasks for subculture after initialization.</li>
	<li>To subculture 3T3 fibroblast cells, remove the flask from the cell culture incubator and wash twice with cold sterile PBS (1x) with gentamycin. Pipet 10 mL of pre-warmed (37 &#176;C water bath) trypsin solution (0.25%) into each T-225 flask.
	<ol>
		<li>Place the flask on a warming plate for 3-8 min. Remove the flask and gently use palms to tap the walls of the flask to detach cells and confirm cell detachment under inverted microscope. Trypsin solution may appear cloudy with detached 3T3 cells.</li>
	</ol>
	</li>
	<li>Pipet the trypsin solution 3&#8211;4x to wash the cell growth surface of the flask and transfer the trypsinized cell to 30 mL of CGM in a 50 mL conical tube. Rewash the flask 3&#8211;5x with 10 mL of the CGM-trypsin-cell mixture from the 50 mL conical tube. 
	NOTE: The contents of two flask contents can be added to a 50 mL tube; if only one flask is used, then place 10 mL of trypsin/cells into 20 mL of CGM in a 50 mL tube.</li>
	<li>Centrifuge the 50 mL conical tube at 160 x g for 7 min at 4 &#176;C.</li>
	<li>Now aspirate the supernatant and resuspend the cell pellet with 5 mL of CGM, pipetting gently ~10x to completely disrupt the cell pellet. Add 10 mL of CGM to bring the total volume of the 50 mL conical tube to 15 mL.
	<ol>
		<li>Mix again ~10x and place 5 mL of the cell suspension into 3 separate T-150 flasks. The subculture ratio is 1:3 in T-225 flasks or 1:4 in T-150 flasks. Add 30 mL of pre-warmed 3T3 CGM to each of the flasks.</li>
	</ol>
	</li>
	<li>Label the flask with the cell line name, the date of cell seeding, and the passage number.</li>
	<li>To freeze down 3T3 cells following centrifugation in the subculture step, remove the 50 mL tube from the centrifuge and place on an ice bucket. Aspirate the supernatant and resuspend the cell pellet with 1 mL of 5% DMSO-DMEM mixture (see Table 1) for each flask harvested.</li>
	<li>Place 1 mL of DMSO-DMEM mixture with cell suspension into each cryovial (one cryovial per 3T3 flask harvested). Label the vial with the passage number, the cell line name (3T3), and the date that the cells were frozen.</li>
	<li>Place these 3T3 cell vials into cell freezer jar (see Table of Materials) and transfer to -70 &#176;C freezer for &#62;5 h. Careful remove and transfer the vials from the cell freezer jar and place in a liquid nitrogen storage tank for long-term storage until next use. Enter information about the cell line name, the location in tank and the date in the laboratory liquid nitrogen inventory sheet.</li>
</ol>
2. Preparation of 3T3 Feeder Layer for X-ray Irradiation and Seeding
<ol>
	<li>Prepare 3T3 cells one week before their irradiation and allow them to grow ~100% confluence. The 3T3 cells are typically within the passages of 120 to 130.</li>
	<li>For successful clonogenic assays of primary keratinocytes, before seeding 3T3 cells, surface coat the 60 mm Petri dishes with a collagen.
	<ol>
		<li>Pipet around 2&#8211;3 mL of the collagen-coating solution into 60 mm Petri dishes to coat the bottom surface and remove the excess collagen-coating liquid (see Table 1). Transfer the Petri dishes to a 37 &#176;C incubator with 5% CO2 for a minimum of 1 h. Do not let the dishes dry in incubator.</li>
	</ol>
	</li>
	<li>X-ray irradiation step: Follow steps 1.4&#8211;1.6. Use 50 mL of fresh CGM to re-suspend the cell pellet, close the conical tube and seal with paraffin sealing film. To avoid any contamination, use plastic bags to double bag the paraffin-sealed cell containing tube and place on ice. A mitomycin based alternative feeder layer can be used for keratinocyte culture7.</li>
	<li>Irradiate fibroblast 3T3 cells in the 50 mL conical tube with a 5,000 rads (50 Cgy) dose with a biological x-ray machine. Irradiate these cells along with the CGM media.
	<ol>
		<li>Following X-ray irradiation, centrifuge cells at 160 x g for 7 min at 4 &#176;C. Aspirate the supernatant media and gently resuspend cell pellet with 5 mL of CGM triturating gently ~10x. Now bring the final volume to 30 mL with additional 25 mL of medium and triturate 10x. These triturating steps are crucial for uniform 3T3 fibroblast layer for clonogenicity assay.</li>
	</ol>
	</li>
	<li>Viable Cell counting: Count the keratinocytes using a regular glass hemocytometer. Take approximately 0.5 mL of the cells and place into a 1.5 mL tube. Remove 200 &#181;L of cell mixture and add an equal volume of 0.4% Trypan blue solution and mixture 3&#8211;4x. Transfer cells with a hemocytometer and count all nucleated cells (small gold and pink cells) as viable cells. Calculate the final concentration of viable gold cells.</li>
	<li>Pipette 1 x 106 3T3 cells (a minimum of 7 x 105) cells may be used) into 60 mm collagen-coated Petri dishes (step 2.2) and gently add 4 mL of modified high-calcium Williams E media (see Table 1). Allow freshly seeded 3T3 cells to attach for 24 h. On the next day, harvest the fresh primary keratinocyte and seed them on top of attached 3T3 fibroblast layer for clonogenic assay as indicated below.</li>
</ol>
3. Primary Keratinocyte Harvesting and Seeding
<ol>
	<li>Euthanize four to five adult female mice via CO2 inhalation according to approved animal facility/IACUC standards. However, this protocol may also be used for single female/male mice.
	<ol>
		<li>Clip approximately 9 cm2 of the dorsal fur with an electric animal clipper and place all clipped mice into a 500 mL jar with enough povidone iodine antiseptic solution (not scrub) to cover them for 1&#8211;2 min. Shake the jar well to evenly coat the antiseptic solution over the mice.</li>
		<li>Pour off the solution and rinse with clean water until the liquid runs clear. Repeat the same antiseptic wash followed by a water rinse. After the antiseptic wash with iodine solution, soak all the mice in 70% ethanol for 5&#8211;10 min. 
		NOTE: Mice with light fur (e.g., BALB/c) will retain a slight yellowish color from the antiseptic iodine washes, while darker mice (e.g., C57BL/6) will not. Notably, cell viability or culture growth is not affected due to the yellow color.</li>
	</ol>
	</li>
	<li>Carefully remove the clipped dorsal skin using thumb forceps and scissors in a laminar flow hood. Place the excised dorsal skin in conical tube filled with PBS/2x gentamycin. Avoid using ventral side skin to prevent mammary cell contamination in culture.</li>
	<li>Using autoclaved forceps and a scalpel, place one dorsal skin at a time hairy side down onto a thin Petri dish. Start scraping all subcutaneous tissue including fat from the ventral side of the skin tissue until it is semi-translucent. Try to remove maximum traces of subcutaneous tissue without tearing the skin (hair follicle part). Also, do not scrape for a long time and avoid skin drying.
	<ol>
		<li>Keep scraped skin in PBS solution until all other remaining skins are processed. Using a scalpel, slice skins into 0.5 cm x 1&#8211;1.5 cm strips and place the hairy side up onto a sterile Petri dish.</li>
	</ol>
	</li>
	<li>Carefully pipette 20 mL of PBS/2x Gentamicin solution mixed with trypsin (0.25%) into a 100 mm x 20 mm plastic Petri dish. Using sterile forceps, transfer the skins and float them hairy side up on the surface of trypsin solution for 2 h in a 32 &#176;C incubator. 
	NOTE: Trypsinization time and temperature are crucial for good yield of highly culturable primary keratinocytes. Although other methods may provide good yields of viable cells, the culturability of the keratinocytes has been less satisfactory.
	<ol>
		<li>During this 2 h incubation time, coat dishes with collagen coating and place them at 37 &#176;C for 1 h (see step 3.1.2). For clonogenic assays, the 60 mm Petri dishes have already been coated 24 h prior to keratinocyte harvesting to allow the irradiated 3T3 feeder layer to attach to bottom and spread evenly. 
		NOTE: Coating of culture dishes for clonogenic culture is very important for proper attachment, colony formation, and ultimate growth/proliferation of mice epidermal keratinocytes.</li>
	</ol>
	</li>
	<li>Epidermal scraping step: Arrange a sterile plastic square Petri dish and create a surface at a 30&#176; incline for proper epidermal scraping with 15 mL of harvesting medium (see Table 1). Carefully remove a floating skin strip from the trypsin solution with curved forceps. With a new scalpel blade, scrape off the epidermis and hairs into the medium.
	<ol>
		<li>Do not use excessive force, but use sufficient force to scrape the epidermis, or it will affect the cell viability.</li>
		<li>During epidermis scraping, keep the blade perpendicular to the skin piece. If the blade is angled toward the motion of the blade, there are more chances of skin tearing. If the blade is angled away from the blade movement, there are more chances of insufficient epidermis removal.</li>
	</ol>
	</li>
	<li>Carefully pour the harvesting medium containing scraped epidermal cells into a sterile 60 mL jar (autoclavable and reusable) with a 1.5 inch magnetic stir bar. Rinse the Petri dish with additional harvesting medium to collect remaining epidermal cells and bring the final volume to 30 mL. Use a magnetic stirrer and stir at 100 rpm for 20 min at room temperature. This process will remove the trapped epidermal cells from hairs.</li>
	<li>Place a sterile 70 &#181;m cell strainer on the top of a 50 mL conical tube. The cell strainer fits on top of a 50 mL conical tube.
	<ol>
		<li>Take the jar inside a biosafety hood. Remove the stir bar and pour the contents into strainer filter attached to a 50 mL conical tube. Strain out undesired hair and sheets of stratum corneum.</li>
		<li>Press the hairs along with stratum corneum materials within the strainer and manipulate them to release the trapped hair cells. Use 5 mL of harvesting medium to allow remaining trapped cells to release and flow into the tube. Bring the total volume up to 50 mL in conical tube.</li>
	</ol>
	</li>
	<li>Cap the 50 mL conical tube with cell filtrate and centrifuge at 160 x g for 7 min at 4 &#176;C. Next aspirate the supernatant and add 5 mL of harvesting medium. Resuspend the cell pellet by triturating gently ~20x with a 5 mL pipet.
	<ol>
		<li>At this step, keep the cells in an icebox to avoid any aggregation. Add 25 mL of harvesting medium and triturate again (20&#8211;25x).</li>
		<li>To ensure an accurate keratinocyte count at this step, take 1 mL of cell suspension and add 19 mL harvesting medium. Now the cell dilution is 1:20 in 50 mL conical tube. If keratinocytes are harvested from a single mouse, adjust the volume of the cell suspension. Instead of 30 mL, use 15 mL and make a 1:10 dilution for counting the cells.</li>
	</ol>
	</li>
	<li>Pipet 0.5 mL of diluted cells from the 50 mL conical tube (1:20 dilution) and transfer to a 1.5 mL autoclaved microcentrifuge tube. Remove 0.2 mL (200 &#181;L) of cell mix to another 1.5 mL microcentrifuge tube and add an equal volume (200 &#181;L) of 0.4% Trypan blue solution. Gently mix this solution 3x and transfer cells to a hemocytometer for counting nucleated keratinocytes.
	<ol>
		<li>Score all dark blue cells positive for Trypan blue as non-viable dead cells, and score small gold and pinkish cells as viable cells. The final keratinocyte yield per mouse should be around 30 million viable cells.</li>
	</ol>
	</li>
	<li>Centrifuge the original 50 mL conical tube (from step 3.8) for 7 min at 160 x g at 4 &#176;C. At this step, resuspend the cells in desired medium and make the required dilution for seeding cells.
	<ol>
		<li>For mass culture, seed 2 to 4 million viable gold cells in each 35 mm Petri dish in cell culture medium (KGM-type medium + supplements for monolayer cultures) (see Table 1). For a clonogenic (colony formation) assay, seed 1,000 cells per 60 mm Petri dish on X-ray irradiated Swiss mouse 3T3 feeder layers with collagen-coating and modified William&#8217;s E medium with supplements and serum.</li>
		<li>Use a total of 2 to 4 mL medium for 35 mm and 60 mm Petri dishes, respectively. Also, formulate DMEM and Williams E medium procured from ATCC with reduced levels of sodium bicarbonate for use at 5% CO2.</li>
	</ol>
	</li>
	<li>Grow the culture cells (mass or clonogenic) at 32 &#176;C, 95% humidity with 5% CO2. Change the media one day after initial seeding for mass culture, and then 3x weekly thereafter. However, for clonal assays, the first medium change is 2 days after cell seeding and 3x weekly thereafter.</li>
	<li>For clonal culture, cultivate cells at two- and four-week intervals. After completion of clonal cultures (2 or 4 weeks), aspirate the medium. Fix the colonies in 10% buffered formalin overnight at room temperature and stain with 0.5% rhodamine B in autoclave water for 1 h. Then, remove the rhodamine B stain with a pipette from the edge of the dishes.
	<ol>
		<li>Rinse the dishes in cold autoclaved water until the dishes run clear. Put the dishes inclined on the lid of the dishes and let them dry for final colony counting. Notably, when performing clonogenic culture assay, never pipet &#60;1 mL of cells. If cells are concentrated, serially dilute the cell concentration.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Morris, R. J., Tacker, K. C., Fischer, S. M., Slaga, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+primary+in+vitro+clonogenic+keratinocytes+from+normal+adult+murine+epidermis,+following+initiation,+and+during+promotion+of+epidermal+tumors.">Quantitation of primary in vitro clonogenic keratinocytes from normal adult murine epidermis, following initiation, and during promotion of epidermal tumors.</a> Cancer Research. 48 (22), 6285-6290 (1988).</li><li>Tani, H., Morris, R. J., Kaur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+for+murine+keratinocyte+stem+cells+based+on+cell+surface+phenotype.">Enrichment for murine keratinocyte stem cells based on cell surface phenotype.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (20), 10960-10965 (2000).</li><li>Morris, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+and+profiling+adult+hair+follicle+stem+cells.">Capturing and profiling adult hair follicle stem cells.</a> Nature Biotechnology. 22 (4), 411-417 (2004).</li><li>Trempus, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+for+living+murine+keratinocytes+from+the+hair+follicle+bulge+with+the+cell+surface+marker+CD34.">Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34.</a> Journal of Investigative Dermatology. 120 (4), 501-511 (2003).</li><li>Redvers, R. P., Kaur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+cultivation+of+primary+adult+murine+keratinocytes.">Serial cultivation of primary adult murine keratinocytes.</a> Methods in Molecular Biology. 289, 15-22 (2005).</li><li>Jensen, K. B., Driskell, R. R., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+proliferation+and+differentiation+capacity+of+stem+cells+using+disaggregated+adult+mouse+epidermis.">Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis.</a> Nature Protocols. 5 (5), 898-911 (2010).</li><li>Blacker, K. L., Williams, M. L., Goldyne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitomycin+C-treated+3T3+fibroblasts+used+as+feeder+layers+for+human+keratinocyte+culture+retain+the+capacity+to+generate+eicosanoids.">Mitomycin C-treated 3T3 fibroblasts used as feeder layers for human keratinocyte culture retain the capacity to generate eicosanoids.</a> Journal of Investigative Dermatology. 89 (6), 536-539 (1987).</li><li>Morris, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Procedure for harvesting epidermal cells from the dorsal epidermis of adult mice for primary cell culture in &#8220;high calcium&#8221; defined medium. , (1994).</li><li>Wu, W. Y., Morris, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+harvest+and+assay+of+in+vitro+clonogenic+keratinocytes+stem+cells+from+mice.">Method for the harvest and assay of in vitro clonogenic keratinocytes stem cells from mice.</a> Methods in Molecular Biology. 289, 79-86 (2005).</li><li>Popova, N. V., Teti, K. A., Wu, K. Q., Morris, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+keratinocyte+stem+cell+regulatory+loci+implicated+in+skin+carcinogenesis.">Identification of two keratinocyte stem cell regulatory loci implicated in skin carcinogenesis.</a> Carcinogenesis. 24 (3), 417-425 (2003).</li><li>Popova, N. V., Tryson, K. A., Wu, K. Q., Morris, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+keratinocyte+colony+number+is+genetically+controlled.">Evidence that the keratinocyte colony number is genetically controlled.</a> Experimental Dermatology. 11 (6), 503-508 (2002).</li><li>Trempus, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+microarray+transcriptome+profiling+of+CD34-enriched+mouse+keratinocyte+stem+cells.">Comprehensive microarray transcriptome profiling of CD34-enriched mouse keratinocyte stem cells.</a> Journal of Investigative Dermatology. 127 (12), 2904-2907 (2007).</li><li>Baer-Dubowska, W., Morris, R. J., Gill, R. D., DiGiovanni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+covalent+DNA+adducts+in+mouse+epidermal+subpopulations+after+topical+application+of+benzo(a)pyrene+and+7,12-dimethylbenz(a)anthracene.">Distribution of covalent DNA adducts in mouse epidermal subpopulations after topical application of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene.</a> Cancer Research. 50 (10), 3048-3054 (1990).</li><li>Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal,+multipotency,+and+the+existence+of+two+cell+populations+within+an+epithelial+stem+cell+niche.">Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche.</a> Cell. 118 (5), 635-648 (2004).</li></ol>

The authors have nothing to disclose.

The keratinocyte harvesting method described here was developed to produce high yields of highly culturable primary epidermal keratinocytes from the back of adult female mice. These keratinocytes are suitable for flow cytometry, molecular biology application, and primary cell culture, including the clonogenic assay reported here. This keratinocyte harvesting method also has been useful for studying other downstream aspects of cutaneous chemical carcinogenesis and tumor promotion1,13.
There are several critical steps in the protocol. First, for rigor and reproducibility, female mice of age 54 &#177; 2 days were used. This has enabled us to perform sensitive and quantitative colony assays from multiple strains of mice and to use them as a phenotype in linkage analysis leading to identification of least one new stem cell regulatory gene10,11. Second, it is helpful to cut the skins into smaller pieces, as the trypsinization is more effective than trying to keep the skin whole. Third, the time and the temperature of the trypsin flotation is very important to the subsequent culturability. Moreover, one needs to &#34;poke considerably&#34; at the hairs remaining on the filter to dislodge the attached cells. This step is important to obtain high yields of cells. Furthermore, the 32 &#176;C temperature of the incubator is important for the longer-term cultivation of keratinocytes from adult mice. Finally, in this protocol, the parallel readouts of in vitro and in vivo experiments1 are based on primary cultures rather than subcultures or cell lines. Notably, if harvesting primary keratinocytes for the first time using this protocol, keep the remaining dermis after scraping to confirm the full removal of hair follicles along with epidermis by histopathological observation.
The principal strength of this protocol for harvesting adult mouse keratinocytes is that it produces high yields of culturable cells that are suitable for many downstream applications. The chief limitation is that some cell surface epitopes may be sensitive to trypsinization such that immunostaining may be compromised. Therefore, when testing a new cell surface antibody, it may be prudent to use a dispase6 or thermolysin5 based method for harvesting for comparison. The significance of this protocol is that it has been rigorously tested, quantified, and applied to such diverse applications as assays for clonogenic stem cells1,9,10,11, for mass cultures in biochemistry8, for FACS sorting in stem cell analysis3,4, for molecular biology3,12, and for ongoing RNA and DNA sequencing.

Mammalian skin covers the entire body and serves a multitude of functions (e.g., a primary physical barrier, temperature regulation, and moisture retention). Over the last five decades, basic research on mouse skin has yielded new information on the structure and function of the skin. The mouse skin model has greatly helped understanding of the basic biology behind the complex molecular mechanisms of chemical or ultraviolet radiation-induced carcinogenesis. These mouse skin models provided a significant contribution towards understanding of human skin diseases and their mitigation. To explore further molecular dissection of the primary keratinocytes in hair follicle developmental biology, stem cell research, carcinogenesis, and genetic exploration of epidermis, it is frequently desirable to isolate and culture primary epithelial keratinocytes from mice skin to use in parallel with in vivo experiments. The motivating factor in developing this method was the need for an in vitro assay for clonogenic epidermal and hair follicle stem cells1,2. There was also an urgent need for single cell suspensions of epidermal cells suitable for clonogenic assays3, fluorescence activated cell sorting, and flow cytometry3,4. The advantages of this method are a robust harvest increase an order of magnitude over other methods5,6, inclusion of the epithelial portion of the hair follicles, and the high culturability of the harvested cells. In the 1980s, there were no known methods that could meet these requirements. In subsequent years, this method was refined to increase cell yield. The principal limitation of this method would be masking of some trypsin sensitive antibodies that would compromise immunoreactivity6.
The mice received husbandry according to the Specific Pathogen Free guidelines. One to five female mice 54 &#177; 2 days of age were obtained. In older mice, the viability of keratinocytes will be reduced due to anagen phase (growth) of the hair cycle. Also, the process of trypsinization is more difficult compared to young mice. The harvesting procedure has been successfully optimized for the thinner skin of female mice compared to male. Male mice are generally not used for keratinocyte harvests as they have a tendency to fight with one another during housing and therefore may leave scratches or wounds on the skin.

<table>
	<tbody>
		<tr>
			<td>0.4% Trypan blue 1X in 0.9% saline</td>
			<td>Life Technologies Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES Buffer</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>1.5-inch magnetic stir bar with pivot ring</td>
			<td>Bel-Art, Wayne</td>
			<td>371101128</td>
			<td>in 2 oz Nalgene jar</td>
		</tr>
		<tr>
			<td>10 mL disposable pipettes</td>
			<td>BD Falcon</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile conical tube</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>150 cm2 (T-150) culture flask</td>
			<td>Corning</td>
			<td>430825</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Screw Cap Micro Tube Conical</td>
			<td>Sarstedt</td>
			<td>72.608</td>
			<td></td>
		</tr>
		<tr>
			<td>2 oz Nalgene jar</td>
			<td>Thermo Fisher Scientific</td>
			<td>2118-0002</td>
			<td>60 mL</td>
		</tr>
		<tr>
			<td>2.5% Trypsin solution 10X</td>
			<td>Life Technologies Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>225 cm2 (T-225) culture flask</td>
			<td>Corning</td>
			<td>431082</td>
			<td></td>
		</tr>
		<tr>
			<td>32 oz Nalgene Square Storage Bottles: PETG</td>
			<td>Thermo Fisher Scientific</td>
			<td>2019-1000</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td>35-mm sterile culturing dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL disposable pipets</td>
			<td>BD Falcon</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile conicle tube</td>
			<td>BD Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>60-mm sterile culturing dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>90-mm diameter Spectra Mesh filter, 74-&#181;m pore size</td>
			<td>Spectrum Laboratories</td>
			<td>145956</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics (penicillin-streptomycin)</td>
			<td>Life Technologies Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine calf serum (BCS)</td>
			<td>Thermo Fisher Scientific Hyclone</td>
			<td>SH30073.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>BD Biosciences</td>
			<td>354331</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 Ram34 FITC conjugated antibody</td>
			<td>BD Pharmingen</td>
			<td>553733</td>
			<td></td>
		</tr>
		<tr>
			<td>CD49f (alpha6-integrin, GOH3) PE conjugated antibody</td>
			<td>BD Pharmingen</td>
			<td>555736</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer, 70-&#181;m sterile</td>
			<td>BD Discovery Labware</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Bovine, Type I, 30mg</td>
			<td>BD Biosciences</td>
			<td>354231</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolCell Economical cell freezing container for 1 or 2 mL cryovials</td>
			<td>Biocison</td>
			<td>BCS-136PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 2mL Polypropylene Cryogenic Vial, Self-Standing with Round Bottom</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Milli-Q water</td>
			<td></td>
			<td></td>
			<td>made fresh; twice distilled reverse osmosis water</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline 1X, Ca2+ and Mg2+ free</td>
			<td>Life Technologies Gibco</td>
			<td>14190-144</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Hyclone</td>
			<td>SH 300070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand 4 oz. Sterile Specimen Container</td>
			<td>Fisher Scientific</td>
			<td>16-320-730</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin 50 mg/mL 10</td>
			<td>Life Technologies Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>KGM</td>
			<td>Lonza</td>
			<td>CC-3103</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene &#8220;Mr. Frosty&#34; cell freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene PC Straight-Side Wide-Mouth Jars, 16oz</td>
			<td>Thermo Fisher Scientific</td>
			<td>2116-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 22 sterile stainless-steel blades</td>
			<td>BD Bard-Parker</td>
			<td>371222</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 4 scalpel handles</td>
			<td>Biomedical Research Instruments</td>
			<td>26-1200</td>
			<td>In a cup with 70% ethanol</td>
		</tr>
		<tr>
			<td>One pair of full-curve eye dressing forceps</td>
			<td>Militex</td>
			<td>18-784</td>
			<td>In a cup with 70% ethanol</td>
		</tr>
		<tr>
			<td>One pair of scissors</td>
			<td>Biomedical Research Instruments</td>
			<td>25-1050</td>
			<td>In a cup with 70% ethanol</td>
		</tr>
		<tr>
			<td>One pair of thumb dressing forceps</td>
			<td>Militex</td>
			<td>6-4</td>
			<td>In a cup with 70% ethanol</td>
		</tr>
		<tr>
			<td>Oster Pro-Cord/Cordless Trimmer no.40 blade</td>
			<td>Jarden Consumer Solutions/Oster</td>
			<td>78997-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri dish 100 mm X 20 mm sterile</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Brand Plain Stemless Glass Funnel</td>
			<td>Corning</td>
			<td>6240-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2akappa FITC isotype control antibody</td>
			<td>BD Pharmingen</td>
			<td>553929</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2akappa PE isotype control antibody</td>
			<td>BD Pharmingen</td>
			<td>555844</td>
			<td></td>
		</tr>
		<tr>
			<td>SFM</td>
			<td>Life Technologies Gibco</td>
			<td>10725-018</td>
			<td></td>
		</tr>
		<tr>
			<td>SMEM</td>
			<td>Life Technologies Gibco</td>
			<td>11380-037</td>
			<td></td>
		</tr>
		<tr>
			<td>SMEM: Ca2+ and Mg2+ free minimal essential medium for suspension culture</td>
			<td>Life Technologies Gibco</td>
			<td>11380-037</td>
			<td></td>
		</tr>
		<tr>
			<td>Square sterile plastic Petri dishes</td>
			<td>BD Falcon</td>
			<td>351112</td>
			<td></td>
		</tr>
		<tr>
			<td>Swiss mouse 3T3 fibroblasts</td>
			<td>ATCC</td>
			<td>CCL-92</td>
			<td>used within 10 passages</td>
		</tr>
		<tr>
			<td>Triadine; 10% Povidone-Iodine Prep Topical Solution, 16oz</td>
			<td>Triad Group</td>
			<td>10-8216</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Plastic Petri dishes, sterile Space Saver 100 X 10</td>
			<td>VWR</td>
			<td>25384-324</td>
			<td></td>
		</tr>
		<tr>
			<td>Williams E Medium</td>
			<td>Life Technologies Gibco</td>
			<td>12551-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziploc bag</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mouse epidermal keratinocytes and their in vitro clonogenic culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

Typically, yields of epidermal keratinocytes per mouse ranging from 20 x 106 to 30 x 106 Trypan blue excluding cells are obtained8,9. The viability ranges from 80%-90%. Typical seeding efficiencies are around 30% attachment at 24 h. Colony formation on irradiated 3T3 feeder layers is around 60 colonies per 1,000 viable cells seeded for C57BL/6 mice, and approximately 30 colonies per 1,000 cells for Swiss-type mice10,11. Typical results from colony formation assays are illustrated in Figure 1. The number of hair follicle stem cells is approximately 9% CD34+/CD49f+ stem cells for C57BL/6 mice12. Typical results from flow cytometry are given in Figure 2.
Growth characteristics of four different media are illustrated in Figure 3. The media tested include KGM-gold, SFM, William&#39;s E medium, and Morris-2 (a reduced calcium medium developed in the Morris laboratory based on Williams E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58701/58701fig1.jpg" /> 
Figure 1: Representative examples from an assay for clonogenic keratinocytes from adult mice. This photograph demonstrates keratinocyte colony formation from CD-1 female mice 54 days of age treated topically with 1) no treatment, 2) acetone followed one week later by acetone twice weekly for two weeks, 3) DMBA followed one week later by acetone twice weekly for two weeks, 4) acetone followed one week later by TPA twice weekly for two weeks, and 5) DMBA followed one week later by TPA twice weekly for two weeks. After their in vivo treatments, keratinocytes were harvested and seeded at 1,000 cells per dish onto feeder layers of irradiated 3T3, and cultured for two weeks, after which the dishes were fixed with buffered formalin and stained with rhodamine B. The dishes in each column are replicates from each mouse treatment group. Note that the number of larger colonies increased upon treatment with DMBA, and the total number of colonies increased TPA treatment of the mice prior to keratinocyte harvest. Abbreviations: DMBA: 7,12-dimethylbenz[a]anthracene; TPA: 12-O-tetradecanoylphorbol-13-acetate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58701/58701fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58701/58701fig2.jpg" /> 
Figure 2: Representative example of flow cytometry of hair follicle stem cells from adult mice. Briefly, the isolated primary keratinocytes were isolated from dorsal mice skin and filtered for cellular debris using cell filter. The isolated keratinocyte suspension was labeled with CD49f or &#945;6-integrin (PE) and CD34 (FITC) antibodies along with live dead dye and other controls. FITC and PE isotype control antibodies (Rat IgG2akappa) were used for compensation purpose (see Table of Materials). Stem cells were selected by means of CD49f (PE channel), which recognized the external component of the hemi-desmosomes found on basal epidermal and hair follicle cells, and CD34 (FITC channel), which recognized the hair follicle stem cells (i.e., CD34-FITC+ and a6-PE+ (upper right column, total 5%)). The right side column shows the different keratinocyte populations namely CD34-FITC only, CD34-FITC and a6-PE cells (double positive stem cell population), a6-PE only, and unstained cells. Note that there are two CD34+ stem cell populations as have been reported6,14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58701/58701fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58701/58701fig3.jpg" /> 
Figure 3: Comparison of four different media on primary cultures of epidermal cells from adult female mice. Primary keratinocytes were isolated from the dorsal mice skin and counted for seeding using four different media (KGM, SFM, Willem&#39;s-E and Morris-2). Equal numbers of keratinocytes were seeded and followed for their general morphology and growth pattern. Note that the proliferating keratinocytes have slightly different morphologies. KGM, SFM, and Morris-2 all of which are reduced calcium media have the most robust growth after fourteen days. In contrast, the cells do not persist when cultured in Williams E medium that has 1.4 mM calcium and a high ratio of potassium to sodium. Surprisingly, Williams E medium with supplements and twenty percent fetal bovine serum supports keratinocyte clonal cultures far better than any other medium tested, probably because the enriched Williams E medium helps the 3T3 feeder cells to &#34;feed&#34; better. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58701/58701fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solutions and Media</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Harvesting Solutions</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% trypsin (5 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (500 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin (2 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS with 2x gentamycin (45 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) with 2x gentamycin</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Solutions</td>
			<td></td>
		</tr>
		<tr>
			<td>Purecol-fibronectin dish-coating solution:</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES (1 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>116 mM CaCl2 (1 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin 1.0 mg/mL (10 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture medium (100 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin (1 mg)</td>
			<td></td>
		</tr>
		<tr>
			<td>Purecol collagen (1 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing solution</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (2 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with 10% BCS and pen-strep</td>
			<td></td>
		</tr>
		<tr>
			<td>Harvesting Medium</td>
			<td>Needs to be without calcium</td>
		</tr>
		<tr>
			<td>2x gentamycin (1 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (50 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>SMEM (500 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Calcium Williams&#39; E Media with:</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF (5 &#181;g/mL) 1 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine 14.5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone (1 mg/mL) 0.5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin 1 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>LinoAcid-BSA (0.1 mg/mL) 0.5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Ess AminoAcids 4 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins 5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin (5mg/mL) 1 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Vit A (1 mg/1,000 &#181;L) 57.5 &#181;L</td>
			<td></td>
		</tr>
		<tr>
			<td>Vit E 1 mg/mL (4&#176;C)&#160; 3.5 &#181;L</td>
			<td></td>
		</tr>
		<tr>
			<td>Vit D2 (10 mg/mL) 50 &#181;L</td>
			<td></td>
		</tr>
		<tr>
			<td>3T3 fibroblast complete growth medium (CGM)</td>
			<td></td>
		</tr>
		<tr>
			<td>BCS (100 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (900 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (10 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>KGM</td>
			<td>Medium used for mass culture is a reduced calcium medium</td>
		</tr>
		<tr>
			<td>Morris 2 medium with the same supplements as Williams&#39; E</td>
			<td>A reduced calcium variant of Williams E with supplements</td>
		</tr>
	</tbody>
</table>
Table 1: Solution and media recipes.

Watch this Scientific Journal Video about Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture at JoVE.com

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

isolation-mouse-epidermal-keratinocytes-their-vitro-clonogenic

Nanyang Technological University

Research

JoVE Journal

Biology

17.1K Views.  University of Minnesota. The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

Video: Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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 and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...