This work was supported by The National Key Research and Development Program of China (2017YFA0104604), The General Program of National Natural Science Foundation of China (81772093), Military Logistics Research Project (AWS17J005), the Key Program of Shandong Province Natural Science Foundation (ZR2019ZD36) and The Key Research and Development Program of Shandong Province (2019GSF108107) to X.W. Guangdong Basic and Applied Basic Research Foundation (2019A1515110833) and The Fundamental Research Funds of Shandong University (2019GN043) to J.M.

The use of adult foreskin tissues in this protocol has been approved by the Human Research Ethics Committee (No.2015120401, date: May 12, 2015).
NOTE: Perform all the following procedures in a sterile environment to prevent contaminations of cells and cultures.
1. Preparations
<ol>
	<li>Gather fresh adult foreskin tissues from circumcision surgeries in 15 mL tubes containing 10 mL of phosphate buffer saline (PBS) and store in 4 &#176;C. 
	NOTE: Separate the tissues as detailed below within 24 h after their excision.</li>
	<li>Prepare reagents and culture medium.
	<ol>
		<li>Prepare 3% P/S (penicillin/streptomycin) as the washing solution. Mix 50 mL of PBS with 1.5 mL of penicillin (100 U/mL) and streptomycin (100 mg/L) (P/S).</li>
		<li>Prepare the termination solution (neutralization solution) for neutralization of enzymatic digestion. Supplement 1% P/S, 10% fetal bovine serum (FBS) into DMEM medium.</li>
		<li>Prepare TIVA medium to culture melanocytes: Ham&#8217;s F12; 10% FBS; 1x P&#8260;S&#8260;glutamine; 50 ng/mL 12-O-tetradecanoyl phorbol-13-acetate (TPA); 1 x 10-4 M 3-isobutyl-1-methyl xanthine (IBMX); 1 &#956;M Na3VO4; 1 x 10-3 M N-6,2&#8242;-O-dibutyryladenosine-3&#8242;,5&#8242;-cyclic monophosphate (dbcAMP).</li>
	</ol>
	</li>
</ol>
2. The conventional method
<ol>
	<li>Skin tissue pretreatment
	<ol>
		<li>Rinse each adult foreskin tissue once with 10 mL of 75% ethanol (alcohol) for 30 s, and then rinse twice with 10 mL of washing solution (3% P/S), for 5 min each. 
		NOTE: Wash foreskin tissues with 10 mL of PBS in the beginning if there is a lot of blood. All washes are prepared in 100 mm cell culture dishes.</li>
		<li>Scrape each foreskin tissue with a surgical blade to remove subcutaneous adipose tissues and loose connective tissues in the cover of a 100 mm cell culture dish. 
		NOTE: Use a scalpel to scrape the fat layers away until only the thin epidermis and the dense dermis remain.</li>
		<li>Transfer each adult foreskin into another 100 mm culture dish with the dermis side down. 
		NOTE: Cut each adult foreskin tissue into 3-4 mm wide strips using a scalpel blade to make the dispase work more efficiently.</li>
		<li>Add 2.5 mg/mL dispase to each 100 mm culture dish with adult foreskin tissues and incubate for 16 to 20 h at 4 &#176;C. 
		NOTE: Each gram of tissue is digested with 10 mL of dispase solution.</li>
	</ol>
	</li>
	<li>Separation of the epidermis from the adult foreskin tissue
	<ol>
		<li>After digestion for 16 to 20 h, separate the epidermis from the dermis using tweezers. Grab one side of the dermal edge of the tissue with one tweezer and the corresponding position of the epidermal part with another tweezer and gently peel off the epidermis.</li>
		<li>Wash the epidermis 3x with washing solution (3% P/S), and then transfer the epidermis into a tube.</li>
		<li>Submerge the epidermis by adding 5 mL of 0.05% trypsin into the tube and incubate in a water bath for 30 min at 37 &#176;C. 
		NOTE: Shake the tube to ensure the epidermis is completely submerged before incubation.</li>
	</ol>
	</li>
	<li>Collection and culture of primary cells
	<ol>
		<li>Add an equal volume of termination solution (10% FBS, 1% P/S in DMEM) to neutralize the trypsin and pipette the solution up and down 10-15 times to separate the epidermal cells.</li>
		<li>Pass the cell suspension into a new 50 mL tube through a 100 &#181;m mesh filter to remove debris.</li>
		<li>Centrifuge at 200 x g for 5 min.</li>
		<li>Remove the supernatant and add 10 mL of inoculation TIVA medium to resuspend the cells.</li>
		<li>Seed the resuspended cells into a 100 mm culture dish.</li>
		<li>Culture in a 37&#176; C incubator with 5% CO2.</li>
		<li>Change the medium every 2 days.</li>
		<li>Passage cells when they reach 80% confluence.</li>
	</ol>
	</li>
</ol>
3. The new method
NOTE: The procedure for tissue preparation and digestion in the new method is the same as described above for the conventional method, the only difference being that the isolated fresh cells are resuspended in 10 mL of TIVA medium containing 10 &#181;M Y-27632.
<ol>
	<li>Two days after seeding, replace the media with normal TIVA medium without Y-27632. After that, the culture conditions are the same between the new and the conventional methods.</li>
</ol>
4. Cell passaging
<ol>
	<li>Remove the TIVA medium and rinse each culture dish twice with PBS.</li>
	<li>Add 2 mL of 0.05% trypsin per 100 mm cell culture dish. 
	NOTE: Shake the dish to ensure adequate contact between digestive enzymes and the bottom of the dish.</li>
	<li>Digest in a 37 &#176;C incubator for 2 min.</li>
	<li>Use a microscope to check the cells, and make sure that most cells have dissociated from the cell culture dish. 
	NOTE: Gently tap the dish to release the cells to round up and float in the solution. Prolong the digestion time if most cells did not suspend after tapping, but no more than 5 more min.</li>
	<li>Transfer the melanocytes into a 15 mL tube after neutralizing the trypsin activity by adding 2 mL of termination solution. Centrifuge at 200 x g for 5 min.</li>
	<li>Resuspend each cell pellet with 10 mL of TIVA medium after slowly removing the supernatant, and then count the number of melanocytes.</li>
	<li>Plate about 1 x 106 melanocytes in 10 mL of TIVA medium per 100 mm cell culture dish.</li>
	<li>Renew the cell culture medium every 48 h.</li>
</ol>

<ol>
	<li>Costin, G. E., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skin+pigmentation:+melanocytes+modulate+skin+color+in+response+to+stress.">Human skin pigmentation: melanocytes modulate skin color in response to stress.</a> FASEB Journal. 21 (4), 976-994 (2007).</li><li>Battyani, Z., Xerri, L., Hassoun, J., Bonerandi, J. J., Grob, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosinase+gene+expression+in+human+tissues.">Tyrosinase gene expression in human tissues.</a> Pigment Cell Research. 6 (6), 400-405 (1993).</li><li>Michaloglou, C., 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=BRAFE600-associated+senescence-like+cell+cycle+arrest+of+human+naevi.">BRAFE600-associated senescence-like cell cycle arrest of human naevi.</a> Nature. 436 (7051), 720-724 (2005).</li><li>Hu, F., Staricco, R. J., Pinkus, H., Fosnaugh, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanocytes+in+tissue+culture.">Human melanocytes in tissue culture.</a> Journal of Investigative Dermatology. 28 (1), 15-32 (1957).</li><li>Eisinger, M., Marko, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+proliferation+of+normal+human+melanocytes+in+vitro+in+the+presence+of+phorbol+ester+and+cholera+toxin.">Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin.</a> Proceedings of the National Academy of Sciences of the United States of America. 79 (6), 2018-2022 (1982).</li><li>Hirobe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+keratinocyte-derived+factors+involved+in+regulating+the+proliferation+and+differentiation+of+mammalian+epidermal+melanocytes.">Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes.</a> Pigment Cell Research. 18 (1), 2-12 (2005).</li><li>Halaban, R., 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=Basic+fibroblast+growth+factor+from+human+keratinocytes+is+a+natural+mitogen+for+melanocytes.">Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes.</a> Journal of Cell Biology. 107 (4), 1611-1619 (1988).</li><li>Kunisada, T., 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=Keratinocyte+expression+of+transgenic+hepatocyte+growth+factor+affects+melanocyte+development,+leading+to+dermal+melanocytosis.">Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis.</a> Mechanisms of Development. 94 (1-2), 67-78 (2000).</li><li>Hirobe, T., Shinpo, T., Higuchi, K., Sano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+human+melanocytes+is+regulated+by+endothelin-1+and+stem+cell+factor+in+synergy+with+cyclic+AMP+and+basic+fibroblast+growth+factor.">Life cycle of human melanocytes is regulated by endothelin-1 and stem cell factor in synergy with cyclic AMP and basic fibroblast growth factor.</a> Journal of Dermatological Science. 57 (2), 123-131 (2010).</li><li>Wen, J., Zu, T., Zhou, Q., Leng, X., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-27632+simplifies+the+isolation+procedure+of+human+primary+epidermal+cells+by+selectively+blocking+focal+adhesion+of+dermal+cells.">Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (2), 1251-1255 (2018).</li><li>Liu, Z., 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=A+simplified+and+efficient+method+to+isolate+primary+human+keratinocytes+from+adult+skin+tissue.">A simplified and efficient method to isolate primary human keratinocytes from adult skin tissue.</a> Journal of Visualized Experiments. (138), e7862 (2018).</li><li>Terunuma, A., Limgala, R. P., Park, C. J., Choudhary, I., Vogel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+procurement+of+epithelial+stem+cells+from+human+tissue+specimens+using+a+Rho-associated+protein+kinase+inhibitor+Y-27632.">Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632.</a> Tissue Engineering Part A. 16 (4), 1363-1368 (2010).</li><li>Cerqueira, M. T., Frias, A. M., Reis, R. L., Marques, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfollicular+epidermal+stem+cells:+boosting+and+rescuing+from+adult+skin.">Interfollicular epidermal stem cells: boosting and rescuing from adult skin.</a> Methods in Molecular Biology. 989, 1-9 (2013).</li><li>Zhou, Q., 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=ROCK+inhibitor+Y-27632+increases+the+cloning+efficiency+of+limbal+stem/progenitor+cells+by+improving+their+adherence+and+ROS-scavenging+capacity.">ROCK inhibitor Y-27632 increases the cloning efficiency of limbal stem/progenitor cells by improving their adherence and ROS-scavenging capacity.</a> Tissue Engineering Part C, Methods. 19 (7), 531-537 (2013).</li><li>Yokoyama, 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=Pharmacologic+suppression+of+MITF+expression+via+HDAC+inhibitors+in+the+melanocyte+lineage.">Pharmacologic suppression of MITF expression via HDAC inhibitors in the melanocyte lineage.</a> Pigment Cell &amp; Melanoma Rresearch. 21 (4), 457-463 (2008).</li><li>Mi, 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=A+ROCK+inhibitor+promotes+keratinocyte+survival+and+paracrine+secretion,+enhancing+establishment+of+primary+human+melanocytes+and+melanocyte-keratinocyte+co-cultures.">A ROCK inhibitor promotes keratinocyte survival and paracrine secretion, enhancing establishment of primary human melanocytes and melanocyte-keratinocyte co-cultures.</a> Pigment Cell &amp; Melanoma Research. 33 (1), 16-19 (2020).</li></ol>

All authors declare no interest of conflict.

The protocol described here was based on a recent publication16. Some attention should be paid to the following critical steps to achieve the best results with the new method. First, successful separation of the epidermis and the dermis is crucial. Cut the adult foreskin tissues into 3-4 mm wide strips using a scalpel blade to make the dispase work more thoroughly and easily to separate the epidermis from the dermis. Second, when separating those two layers, contamination of the dermis should be avoided since dermal fibroblasts can also grow in melanocyte culture medium. Third, the trypsin digestion step should be less than 30 min; otherwise, it will significantly decrease the viability of the isolated cells.
This new method significantly shortens the time needed for melanocyte isolation. Human epidermal melanocytes produce melanin, which protects the skin from solar irradiation damage. Eisinger and Marko proposed the &#64257;rst practical method for the culture of human melanocytes from the epidermis5. Most importantly, the tissues used usually come from newborn babies and it is very difficult to isolate primary melanocytes from adult skin tissues. The Rho-associated protein kinase inhibitor Y-27632 significantly improves the efficiency of epidermal stem cell isolation11-13. Additionally, we also reported a method for isolating human primary epidermal cells from skin tissues in the presence of Y-2763210,11. In this protocol, Y-27632 was shown to efficiently increase the yield of primary melanocytes. Using the conventional method has a low cell recovery rate and needs around 3 to 4 weeks of culture for melanocytes to grow in sufficient numbers to passage. This new method, in which Y-27632 is added to the TIVA growth medium, increased melanocyte yield by about five times, and the melanocytes obtained can proliferate normally. We also tested the new method using a commercial medium and found similar results. Regarding the mechanism involved, a recent study demonstrated that the effect of Y-27632 to enhance the efficiency of melanocyte isolation occurs mainly through keratinocytes16.
Importantly, we obtained pure melanocytes with normal function using the new method. The Passage 1 (P1) melanocytes were nearly all stained for MITF expression indicating that pure melanocytes are obtained after the first passage. Primary melanocytes with high growth potential are very important for biological research and clinical applications. The melanocytes isolated using the new method can be induced to pigment after treatment with forskolin, an activator of adenylate cyclase that increases cyclic AMP levels, indicating these melanocytes can function normally, which will be useful for studying pigmentation defects and melanoma14.
In summary, a highly efficient method was successfully established to isolate primary melanocytes from adult skin tissues. This improved method could contribute to provide sufficient numbers of melanocytes in a rapid manner for biological research and for clinical applications.

The goal of this study was to develop a simple and effective protocol to isolate melanocytes from the epidermis of adult skin for biological research and clinical applications. Melanocytes, which are located in the basal epidermis of the skin and in hair follicles, play an important role in pigmentation of the skin and hair by producing melanin1. The resulting skin pigmentation from epidermal melanocytes acts as an ultraviolet radiation &#64257;lter that reduces/prevents DNA damage to underlying cells in the skin2. The abnormal proliferation of melanocytes in the skin is quite common, such as in the formation of benign nevi (moles) in which melanocytes potentially transform to oncogenic growth followed by cellular senescence3.
Since 1957, the isolation and subsequent culture of human primary melanocytes has been possible4, but only since 1982 has there been an efficient method that can reproducibly establish cultures of human melanocytes from the epidermis5. The conventional method to isolate primary melanocytes from the epidermis involves a two-step enzymatic digestion. Briefly, the skin is initially digested with dispase to separate the epidermis from the dermis, after which the epidermis is digested with trypsin to produce suspensions containing melanocytes and keratinocytes, which can then be selectively grown in different media. Currently, the initial culture usually takes about 3 to 4 weeks for melanocytes to reach confluency using the conventional method, which is likely due to the low efficiency of their isolation. Therefore, increasing the initial production of melanocytes from adult skin tissues would be quite helpful for both laboratory research and clinical applications.
Many growth factors, most of which have been shown to be secreted by keratinocytes (e.g., &#945;-MSH, ACTH, bFGF, NGF, ET-1, GM-CSF, HGF, LIF and SCF)5-8, regulate the differentiation and proliferation of mammalian melanocytes. In the absence of feeder layers, the lack of those growth factors leads to the decreased proliferation of melanocytes and their increased apoptosis9. The co-culture of keratinocytes as feeder cells and melanocytes could lead to accelerated melanocyte proliferation and reduced apoptosis. However, the co-culture method not only demands more skin tissues to prepare keratinocytes, which is not practical, but also could not work efficiently because the culture conditions required by melanocytes does not favor keratinocyte growth, and vice-versa.
Previous studies have reported that the addition of Y-27632, a ROCK inhibitor, into the growth medium can enhance the yield of human primary epidermal cells from skin tissues10-14. Therefore, it would be interesting to test whether the isolation of human primary melanocytes would benefit from the presence of Y-27632. Indeed, the addition of Y-27632 into the inoculation TIVA medium15, which contains TPA, IBMX, Na3VO4 and dbcAMP, significantly increased the yield of primary melanocytes isolated from foreskin tissues in the initial cultures16. Compared to the conventional protocol, this new protocol is significantly more efficient in increasing the yield of melanocytes16. Therefore, this protocol describes the new method in detail using adult skin tissues based on a previous study16 in order to promote its application in basic and applied biological research.

<table>
	<tbody>
		<tr>
			<td>Alexa fluor-594 donkey anti-mouse IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21203</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Alexa fluor-594 donkey anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37119</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning incorporated</td>
			<td>431792</td>
			<td>Cell filtration</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifuge</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubating</td>
		</tr>
		<tr>
			<td>Constant Temperature Shaker</td>
			<td>Shanghai Boxun</td>
			<td>150036</td>
			<td>For water bath</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abcam</td>
			<td>ab104139</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For melanocyte isolation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>C11995500</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1AC5</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>5E44316</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>MCE</td>
			<td>HY-15371</td>
			<td>Induce pigmentation</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Thermo Scientific</td>
			<td>11330032</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>5C42258</td>
			<td>For cell microscopic observation</td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methyl xanthine (IBMX)</td>
			<td>Sigma</td>
			<td>17018</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>mouse anti-human MITF</td>
			<td>Abcam</td>
			<td>ab12039</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>melanocytes culture medium</td>
		</tr>
		<tr>
			<td>N6,2&#39;-O-dibutyryladeno-sine 3&#39;,5&#39;-cyclic monophosphate (dbcAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>12-O-tetradecanoyl phorbol-13-acetate (TPA&#65289;</td>
			<td>Sigma</td>
			<td>79346</td>
			<td>melanocyte culture medium</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>rabbit anti-human Ki-67</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>For Immunofluorescence</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Solarbio Life Science</td>
			<td>P1020-500</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifugation</td>
		</tr>
		<tr>
			<td>TC20TM automated cell counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td>Automatic cell counting</td>
		</tr>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td>For melanocyte dissociation</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>For melanocyte isolation</td>
		</tr>
	</tbody>
</table>

y-27632 enriches the yield of human melanocytes from adult skin tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

Figure 1 shows a schematic diagram comparing the conventional and the new methods. The procedure for tissue preparation and digestion with the new method is the same as the procedure for the conventional method, the only difference being that the isolated cells are resuspended in 10 mL of TIVA medium in the presence of 10 &#181;M Y-27632. Two days after seeding, replace the media with normal TIVA medium without Y-27632. The conventional method of isolating primary melanocytes from skin tissues usually requires about 3 to 4 weeks for melanocytes to grow in sufficient numbers to passage, but more importantly, the tissues usually come from newborn babies and it is very difficult to isolate primary melanocytes from adult skin tissues. However, using the new method, melanocytes from adult tissues are observed in significantly greater numbers than melanocytes isolated using the conventional method. Microscopic views were taken at days 3, 6 and 8 when isolating the melanocytes from adult skin tissue using the conventional method and the new method (Figure 2A). In these microscopic views, the new method significantly increased the yield of melanocytes at days 6 and 8. Melanocyte numbers were then quantified by counting at least 10 different microscopic fields at days 3, 6 and 8. The results showed that the number of melanocytes isolated using the new method is much higher than the number of melanocytes isolated using the conventional method at days 6 and 8 (Figure 2B). After 8 days of culture, the melanocytes were harvested using trypsin and then counted with an automated cell counter in each single 100 mm dish, which revealed that the new method increased the melanocyte yield by about five times (Figure 2C). Microscopic views taken at day 9, one day after passaging, showed that the passaged melanocytes proliferated normally (Figure 2D), which was confirmed by Ki67 staining (Figure 2E).
The enhanced yield of melanocytes by Y-27632 during the initial culture after isolation has been shown through the regulation of keratinocytes in a previous publication16. As shown in Figure 3, Y-27632 did not increase the growth and proliferation of passaged melanocytes (Figure 3A-B) and did not inhibit the apoptosis of melanocytes (Figure 3C-D). However, Y-27632 significantly increased keratinocyte attachment (Figure 3E-H) and also promoted its surviving (Figure 3I) in the melanocyte-culture TIVA media. Either co-culture with keratinocytes in the presence of Y-27632 or the conditioned medium derived from Y-27632 treated keratinocytes could significantly enhance the growth of melanocytes (Figure 3J).
To further characterize the melanocytes isolated using the new method, MITF, a specific melanocyte marker, was analyzed using immunofluorescence (IF) staining to check the purity of melanocytes after initial passaging. Figure 4A shows that the percentage of cells expressing MITF at passage 1 was nearly 100%, indicating that pure melanocytes were obtained after the first passage by the new method, which is similar to the conventional method. To test whether melanocytes isolated using the new method can function normally, they were treated with forskolin (FSK) for 2 days in vitro, which induced the levels of pigmentation of melanocytes (Figure 4B). Taken together, these data suggest that the melanocytes isolated using the new method can function normally and can produce pigment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61226/61226fig01.jpg" /> 
Figure 1: Comparison of the new method and the conventional method.&#160;This scheme shows a comparison of the conventional method with the new isolation method. The only difference is that the isolated fresh cells are resuspended in 10 mL of TIVA medium in the presence of 10 &#181;M Y-27632. Two days after seeding, the medium is replaced with normal TIVA medium without Y-27632. The new method usually achieves a density of melanocytes suitable for passaging at around day 8, while the conventional method usually takes about 20 days to grow sufficient numbers of melanocytes for passaging. <a href="https://www.jove.com/files/ftp_upload/61226/61226fig01large.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/61226/61226fig02.jpg" /> 
Figure 2: The new isolation method increases the production of primary melanocytes.&#160;(A) Representative images of melanocytes prepared by the conventional method (top row) and by the new method (bottom row) at days 3, 6 and 8 after the initial inoculation. (B) Quantification of melanocyte numbers prepared using the conventional method and the new method by counting melanocyte numbers in at least 10 different microscopic fields (cell number/vision) at days 3, 6 and 8. All experiments were performed in triplicate and results are expressed as the average number of cells per microscopic field. (C) After culture for 8 days, melanocytes were harvested using trypsin and the number of melanocytes prepared using the conventional method and the new method were counted using a cell counting plate. The number of cells was calculated from triplicate experiments, and the results show the average number of cells per 100 mm dish. (D) After counting the number of melanocytes using an automated cell counter, melanocytes prepared using the conventional method or the new method were seeded in new dishes and microscopic photos were taken at day 9. (E) Melanocytes isolated using the new method at passage 0 or at passage 1 were fixed and stained with Ki67 antibody (red) and DAPI (nuclei, blue). (B-C), Student&#8217;s t-test was used to analyze differences between the new and the conventional method, ** P &#60; 0.01, ***P &#60; 0.005. All scale bars represent 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61226/61226fig02large.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/61226/61226fig03v5.jpg" /> 
Figure 3: Y-27632 enhances melanocyte growth through regulation of keratinocytes.&#160;(A) Pure melanocytes (passage 3) were treated with 10 &#956;M Y-27632 for 12, 24, 48, and 60 h and the number of melanocytes was analyzed by CCK-8 assay. (B) Pure melanocytes (passage 3) were treated with 10 &#956;M Y-27632 for 48h and then were fixed for Ki67 staining. Quantification of percentage of Ki67 positive cells among total live cells indicated with DAPI nuclear staining. (C-D) Pure melanocytes (passage 3) were treated with 10 &#956;M Y-27632 for 48 h and then were either stained for apoptotic cells with Annexin V-FITC and propidium iodide followed by FACS analysis (C) or fixed for TUNEL assay. (E) Isolated epidermis after trypsin digestion was plated with TIVA medium with or without Y-27632 (10 &#956;M) and 2 days later immunofluorescence analysis of keratinocytes was performed with a pan-cytokeratin antibody (pan-ck). (F) Quantification of keratinocytes in the Y-27632-treated and control dishes from (E) as percentage of keratinocytes in a total of 500 cells counted. (G) Pure passage 3 keratinocytes were seeded in TIVA medium with or without Y-27632 (10 &#956;M). Images of epidermal cells are shown at one day after seeding. (H) Quantification of attached keratinocytes in the Y-27632-treated and control dishes from (G) as the percentage of attached cells in a total of 5000 cells seeded. (I) Pure passage 3 keratinocytes were plated with TIVA medium in the presence or absence of Y-27632. The microscopic images were obtained at 24 h. (J) Left panel: Equal numbers of pure passage 3 melanocytes were plated with TIVA medium in 4 groups with addition(s) as indicated: 1) Y-27632 (10 &#181;M) alone (Y in graph), 2) passage 3 keratinocytes alone , 3) keratinocytes and Y-27632 (10 &#181;M) (K+Y), and 4) no addition (negative control, con). Relative fold changes in melanocyte proliferation compared to the negative control were determined by counting the number of melanocytes at 24 h and 48 h after plating. Right panel: Conditioned TIVA media were obtained from the 4 groups of passage 3 melanocyte cultures in (left panel) at 48 h after plating. Then, equal numbers of passage 3 melanocytes were plated in 4 groups, each with one of the conditioned media. Relative fold changes in melanocyte proliferation compared to the negative control were determined by counting the number of melanocytes at 24 h and 48 h after plating. (A-J) All experiments have been repeated for 3 times, *p&#60;0.05, **p&#60;0.01, ***p&#60;0.005. All Bars in the images represent 100 &#181;m. The figure has been modified from Mi et al.16. <a href="https://www.jove.com/files/ftp_upload/61226/61226fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61226/61226fig04.jpg" /> 
Figure 4: Characterization of melanocytes isolated using the new method.&#160;(A) Representative images of immunofluorescence staining of MITF (red) in melanocytes prepared using the new or the conventional method. DAPI stains nuclei (blue). (B) Cell pellets were collected from melanocytes prepared using the new method and were treated for 2 days with forskolin (FSK) or DMSO vehicle as a control (con). All scale bars represent 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61226/61226fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues at JoVE.com

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

y-27632-enriches-yield-human-melanocytes-from-adult-skin

Universidad San Sebastian

Research

JoVE Journal

Biology

5.4K Views.  Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration. This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Video: Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

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 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 ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...