The authors thank the Damon Runyon Foundation (Innovation Award #38-16 to C.E.B.) and Pelotonia (B.M.M.) for financial support. We are grateful to C. Haines and C. Wormsbaecher who provided comments to improve the manuscript text. This work benefitted from the Ohio State Comprehensive Cancer Center&#8217;s Analytical Cytometry Shared Resource which is supported by NIH P30 CA016058.

Obtain approval from your institutional animal ethics committee before commencing this or any other study involving animals. Experiments performed in this protocol were approved by the Ohio State University&#8217;s Institutional Animal Care and Use Committee (IACUC, protocol #2012A00000134).
1. Protocol Preparation
NOTE: The following reagent preparation instructions are appropriate for the generation of 9 cm2 melanocyte and fibroblast cultures from a single mouse. Refer to the reagent preparation guide in Table 1 for larger scale isolations.
<ol>
	<li>Prepare 1.5 mL of Collagen Solution containing 50 &#181;g/mL collagen in 0.1 M glacial acetic acid.</li>
	<li>In a laminar flow cabinet, coat one well of a 6-well cell culture dish with 8 &#181;g/cm2 (1.44 mL) Collagen Solution. Ensure that the well is completely covered by the Collagen Solution. Incubate the dish at 37 &#176;C for 3 h or at 4 &#176;C overnight. Prior to use, wash the collagen-coated well twice with 1.35 mL of sterile phosphate buffered saline (PBS) (150 &#181;L of PBS per 1 cm2).</li>
	<li>Assemble the tissue chopper in a laminar flow cabinet by carefully placing a chopping disk on the tissue chopper platform and securing a sterile blade to the movable arm.</li>
	<li>Prepare 3 mL of 1x Antibiotic/Antimycotic Solution by diluting 100x antibiotic/antimycotic stock solution in sterile PBS.</li>
	<li>Prepare 3 mL of fresh Skin Digestion Buffer containing 10% fetal bovine serum, 1% penicillin/streptomycin solution, 1% L-glutamine, 10 mg/mL collagenase type I, 0.25% porcine trypsin and 0.02 mg/mL deoxyribonuclease I in RPMI 1640.</li>
	<li>Prepare 6 mL of fresh Melanocyte Media containing 10% fetal bovine serum, 7% horse serum, 1% penicillin/streptomycin solution, 1% L-glutamine, 0.5 mM di-butyryl cyclic AMP (dbcAMP), 20 nM tetradecanoylphorbol 13-acetate (TPA) and 200 pM cholera toxin (CT) in Nutrient Mix F-12 Ham&#39;s media. 
	NOTE: Concentrated dbcAMP, TPA and CT stock solutions can be made, aliquoted and stored for &#62;1 year at -80 &#176;C. Base media lacking these components can be stored for up to 1 month at 4 &#176;C.</li>
	<li>Prepare 4 mL of Fibroblast Media containing 10% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine in Dulbecco&#8217;s Modified Eagle Medium. This media can be stored at 4 &#176;C for up to 1 month.</li>
	<li>Prepare 40 mL of 4% Paraformaldehyde Solution by diluting 16% paraformaldehyde in PBS. Store any excess at 4 &#176;C for 1 month or -20 &#176;C for up to 1 year.</li>
	<li>Prepare a 1% saponin stock solution by mixing 0.5 g of saponin in 50 mL of PBS at 37 &#176;C until the saponin has completely dissolved. Sterilize the solution for long-term storage using a 50 mL syringe fitted with a 0.2 &#181;m PES filter. The resulting saponin stock solution can be kept at 4 &#176;C for 1 month.</li>
	<li>Prepare 200 &#181;L of 0.1% Saponin Solution per mouse by diluting 1% saponin stock solution in PBS containing 3% bovine serum albumin (BSA).</li>
	<li>Prepare 1 mL of Viability Solution by diluting 1 &#181;L of fixable viability dye in 1 mL of PBS.</li>
</ol>
2. Melanocyte and Fibroblast Isolation
<ol>
	<li>Euthanize 0 to 4 day-old male and/or female C57Bl/6J pups by decapitation and remove extremities from the euthanized mice using surgical scissors.</li>
	<li>In a laminar flow cabinet, briefly roll the trunk of each mouse in a sterile Petri dish containing 70% ethanol. Remove the trunk from the ethanol and place it into an empty, sterile Petri dish.</li>
	<li>Using surgical scissors, sterilized in 70% ethanol, make an incision in the skin on the ventral side of the trunk starting from the neck to the tail. Peel the skin from the trunk of the mouse using sterile forceps.</li>
	<li>Place the skin dermis side down in a 6-well dish containing 3 mL of 1x Antibiotic/ Antimycotic Solution and incubate at room temperature for 2-3 min.</li>
	<li>Turn on the tissue chopper with the following settings in place: Slice thickness: 1 &#181;m; Blade force: ~60% of the maximum; Speed: ~50% of the maximum.</li>
	<li>Transfer the skin, dermis side down, to a sterile tissue chopper disk and pass the skin completely through the activated tissue chopper 3 times.</li>
	<li>Transfer the homogenized skin to a sterile, 15 mL conical tube containing 3 mL of Skin Digestion Buffer. Mix the resulting suspension by pipetting up and down 10-15 times with a P1000 micropipette.</li>
	<li>Cap the conical tube and incubate the sample in a 37 &#176;C water bath for 15 min, inverting the tube every 3-5 min.</li>
	<li>Pellet the cells in the skin homogenate by centrifuging the conical tube in a swinging bucket rotor at 750 x g for 5 min at room temperature.</li>
	<li>Using a P1000 micropipette, slowly and completely remove the Skin Digestion Buffer being careful not to disturb the pellet. 
	NOTE: A portion of whole skin can be saved at this stage and used as a control for Step 3: Confirmation of Cellular Purity. Strain these cells through a 70 &#956;m cell strainer and then begin at step 3.5 for further processing.</li>
	<li>Thoroughly resuspend the cell pellet in 1 mL of Melanocyte Media by pipetting up and down 15-20 times with a P1000 micropipette. Add the resulting cell solution dropwise to an uncoated well of a 6-well dish containing 1 mL of Melanocyte Media.</li>
	<li>Place the plated skin homogenate in a tissue culture incubator set at 37 &#176;C and 5% CO2. Allow the cultures to incubate for 40 min. 
	NOTE: During this time, some fibroblasts in the skin homogenate will adhere to the uncoated dish while the melanocytes and keratinocytes remain in suspension.</li>
	<li>Transfer the culture supernatant from the uncoated dish to one well of a pre-washed, collagen-coated 6-well dish. Add G418 to the media such that the final concentration is 100 ng/mL.</li>
	<li>Add 2 mL of Fibroblast Media to one well of the uncoated dish, now containing adherent fibroblasts.</li>
	<li>Incubate both cultures overnight in a tissue culture incubator set at 37 &#176;C and 5% CO2.</li>
	<li>Separately aspirate the media and any debris from each culture, 16-24 h after plating. Wash each dish twice with 1 mL of sterile PBS, and then add 2 mL of fresh Melanocyte Media plus 100 ng/mL G418 to the melanocyte culture and 2 mL of fresh Fibroblast Media to the fibroblast culture.</li>
	<li>After the melanocyte cultures have been treated with G418 for 48 h, wash the cells twice with 1 mL of sterile PBS and add 2 mL of fresh Melanocyte Media without G418 to the culture. 
	NOTE: As fibroblasts in the melanocyte culture continue to die off post-G418 treatment, wash the dish with sterile PBS to remove dead cells and add fresh Melanocyte Media. Melanocyte and fibroblast cultures should be passaged when they reach 70-80% confluency.</li>
</ol>
3. Confirmation of Cellular Purity
<ol>
	<li>Aspirate the media from each culture and carefully wash each dish with 1 mL of sterile PBS.</li>
	<li>Add 0.7 mL of 0.25% trypsin to each culture and incubate the cultures in trypsin at 37 &#176;C and 5% CO2 for 1 min.</li>
	<li>Dislodge the cells by pipetting the trypsin against the bottom of the dish using a P1000 micropipette.</li>
	<li>Add 0.7 mL of the appropriate media to each trypsinized culture and transfer the cell solution into a 1.5 mL microcentrifuge tube.</li>
	<li>Pellet the cells by centrifugation at 750 x g for 2 min at room temperature. Carefully remove and discard the supernatant using a P1000 micropipette. 
	NOTE: Steps 3.1-3.5 can be used to passage melanocyte and fibroblast cultures. The resulting pellet should be resuspended in the appropriate media and placed in a new, uncoated (fibroblasts) or pre-washed, collagen-coated (melanocytes) dish.</li>
	<li>Resuspend the cell pellet in 0.5 mL of ice-cold PBS.</li>
	<li>Enumerate and transfer 0.5 million cells to a pre-chilled 1.5 mL microcentrifuge tube.</li>
	<li>Repeat step 3.5 and then resuspend the cells in 100 &#181;L of Viability Solution. Incubate the cell suspension for 30 min at 4 &#176;C in the dark.</li>
	<li>Repeat steps 3.5-3.6 twice.</li>
	<li>Repeat step 3.5, then fix the cells by resuspending the pellet in 100 &#181;L of ice cold 4% Paraformaldehyde Solution. Incubate the suspension for 15 min at room temperature in the dark.</li>
	<li>Repeat step 3.5 twice, each time resuspending the pellet in 0.5 mL of 3% BSA in 1x PBS.</li>
	<li>Repeat step 3.5, then resuspend the cell pellet in 100 &#181;L of 0.1% Saponin Solution. Incubate the suspension for 15 min at room temperature in the dark.</li>
	<li>Repeat step 3.5, then resuspend the cell pellet in 100 &#181;L of 0.1% Saponin Solution containing 0.5 &#181;g of rabbit anti-gp100 antibody, 0.5 &#181;g of rabbit anti-Fibroblast-specific protein 1 (FSP1) antibody and 0.025 &#181;g of mouse anti-Cytokeratin 14 (K14) antibody. Incubate the suspension for 1 h at room temperature in the dark. 
	NOTE: While the K14 antibody was purchased pre-conjugated to Alexa Fluor 647, the gp100 and FSP1 antibodies were conjugated to CF 555 and CF 488, respectively. The staining of control populations should also commence at this step. Control cell populations should be isolated from culture and processed as described in steps 3.5-3.12.</li>
	<li>Repeat step 3.11 twice, to remove any unbound antibody.</li>
	<li>Repeat step 3.5, then resuspend the cell pellet in 200-400 &#181;L of 3% BSA in 1x PBS.</li>
	<li>Pass the cell solution through a 40 &#181;m cell strainer into a 5 mL polystyrene round-bottom tube and analyze the strained cells by flow cytometry.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocytes+and+their+diseases.">Melanocytes and their diseases.</a> Cold Spring Harbor Perspectives in Medicine. 4 (5), (2014).</li><li>Brenner, M., 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=The+protective+role+of+melanin+against+UV+damage+in+human+skin.">The protective role of melanin against UV damage in human skin.</a> Photochemistry and Photobiology. 84 (3), 539-549 (2008).</li><li>Thody, A. 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=Pheomelanin+as+well+as+eumelanin+is+present+in+human+epidermis.">Pheomelanin as well as eumelanin is present in human epidermis.</a> The Journal of Investigative Dermatology. 97 (2), 340-344 (1991).</li><li>Swope, V. B., Abdel-Malek, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MC1R:+Front+and+Center+in+the+Bright+Side+of+Dark+Eumelanin+and+DNA+Repair.">MC1R: Front and Center in the Bright Side of Dark Eumelanin and DNA Repair.</a> International Journal of Molecular Science. 19 (9), (2018).</li><li>Barsh, G. 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The authors have no conflicts of interest to disclose.

The in vitro culture of primary melanocytes and fibroblasts has led to significant advancements in our understanding of skin biology and disease. This protocol improves upon prior melanocyte isolation methods by reducing the time and technical savvy needed to generate consistent melanocyte cultures while allowing for the simultaneous isolation of skin fibroblasts. A novel, time-saving element of this procedure is that the dermis and epidermis do not need to be separated. Instead, skin cells are isolated using the consist...

Mammalian skin is a multilayered organ that protects the body from foreign pathogens and ultra violet irradiation (UVR). The skin also plays a critical role in homeostatic processes such as wound healing, temperature regulation and vitamin D production1,2,3. Mammalian skin consists of three major cell types: melanocytes, fibroblasts and keratinocytes. These cell types populate different layers of the skin, with keratinocytes making up the epidermis, fibroblasts residing in the dermis and melanocytes localizing to the epidermal-dermal junction and hair follicles4. Here, we detail a simple procedure that enables the concurrent generation of primary melanocyte and fibroblast cultures from murine skin.
Melanocytes are pigment-producing cells found in many locations throughout the human body, including the basal epidermis, iris, cochlea, brain and hair follicles5. The primary function of melanocytes is to generate and secrete melanin-containing vesicles called melanosomes5,6. Melanosomes contain two major classes of melanin: brown/black eumelanin and yellow/red pheomelanin6,7. Biochemical processes within the melanocyte regulate the relative abundance of each melanin species and help to determine hair, skin and eye color8,9. Melanin also serves to absorb UVR and protect sun-exposed tissues from mutagenesis10.
Melanocyte dysfunction can cause pigmentary defects and increase skin cancer susceptibility. For example, the hyper-pigmented skin patches characteristic of melasma are the result of focal melanin overproduction, whereas germline genetic mutations which compromise genes involved in melanin synthesis lead to albinism11,12. Intimate knowledge of melanocyte biology is required to develop strategies that will correct such pigmentary defects and ultimately improve the psychosocial well-being of individuals afflicted with these diseases. Deficits in melanin production and/or the preferential synthesis of pheomelanin are also associated with increased skin cancer risk10. This risk is believed to result from reduced UVR protection6,13. Thus, methods to enhance or restore eumelanin production in melanocytes may reduce the incidence of skin cancer in these populations.
Mesenchymal fibroblasts establish the connective tissue and structural support for all organs of the body, including the dermal layer of the skin14. Excretion of proteins such as collagen, elastin, laminin and fibronectin enable fibroblasts to form the extracellular matrix (ECM) that is essential for tissue integrity1,14. Fibroblasts also play essential roles in processes such as wound healing, inflammation, angiogenesis and cancer formation/progression1,15,16.
Similar to melanocytes, defects in fibroblast activation and function can promote tumorigenesis and disease. For example, inappropriate fibroblast activation commonly leads to the formation of fibrosis, resulting from the enhanced deposition of excess ECM components into the surrounding tissue. As fibroblasts maintain much of the body&#8217;s structural integrity, fibrosis promotes diseases that affect numerous tissues and organs, including idiopathic pulmonary fibrosis, systemic sclerosis, liver cirrhosis and cardiac fibrosis15. Fibroblasts also play a critical role in cancer16. Cancer associated fibroblasts (CAFs) are the most abundant non-malignant cells in the microenvironment of many tumors. CAFs have been shown to promote tumor proliferation, progression and therapeutic resistance by modulating tissue stiffness, local cytokine production and immune function16.
Primary cell cultures provide researchers with genetically tractable models to identify and mitigate melanocyte and fibroblast defects that lead to disease. However, current methods to establish melanocyte cultures are time consuming and technically challenging. The need for trypsinization and delicate separation of the epidermis and dermis contributes to variability in experimental yield and makes it difficult to perform large-scale experiments. Furthermore, protocols to simultaneously isolate melanocytes and fibroblasts from whole skin are currently lacking in the field.
We have developed a method to reduce the processing steps, variability and time required to establish melanocyte and fibroblast cultures from the same whole skin sample. Using a mechanical homogenization method followed by a brief digestion, our strategy significantly decreases the amount of hands-on time required to isolate primary melanocytes while enabling concurrent fibroblast isolation. The increased speed, efficiency and consistency of this protocol, in combination with the ability to isolate melanocytes from 0-4 day old mice, provides researchers the flexibility to perform a wider array of experiments than previously possible.

<table border="0" cellpadding="0" cellspacing="0" style="width:860px;" width="859">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">0.2 &#181;m PES sterile syringe filter</td>
			<td style="width:183px;">VWR</td>
			<td style="width:151px;">28145-501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">10 cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:151px;">430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">40 &#181;m cell strainer</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">22363547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">5 mL polystyrene round-bottom tubes</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">352008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">6-well cell culture dish</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">SIAL0516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">70 &#181;m cell strainer</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Antibiotic Antimycotic Solution (100x)</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">A5955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bovine Serum Albumin</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">BP9706-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CF 488A Mix-n-Stain Antibody Labeling Kit</td>
			<td>Biotium</td>
			<td>92273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CF 555 Mix-n-Stain Antibody Labeling Kit</td>
			<td>Biotium</td>
			<td>92274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cholera Toxin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">C8052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Collagen from rat tail</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">C7661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Collagenase Type I</td>
			<td style="width:183px;">Worthington Biochemicals</td>
			<td style="width:151px;">LS004156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Corning Penicillin/Streptomycin Solution</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">30-002-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cytokeratin 14 Antibody Alexa Fluor 647</td>
			<td style="width:183px;">Novus Biologicals</td>
			<td style="width:151px;">NBP2-34403AF647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Deoxyribonuclease I</td>
			<td style="width:183px;">Worthington Biochemicals</td>
			<td style="width:151px;">LS002058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Di-butyryl cyclic AMP</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">D0627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:151px;">12800-082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">eBioscience Fixable Viability Dye eFluor 780</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:151px;">65-0865-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ethanol, 200 proof</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">22032601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Fetal Bovine Serum</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">12306C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">FSP1/S100A4 antibody</td>
			<td style="width:183px;">Millipore Sigma</td>
			<td style="width:151px;">07-2274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">G418 Disulfide</td>
			<td style="width:183px;">P212121</td>
			<td style="width:151px;">LGB-418-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glacial Acetic Acid</td>
			<td style="width:183px;">VWR</td>
			<td style="width:151px;">VWRV0714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Horse Serum</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">26050088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HyClone L-Glutamine</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">SH3003402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">McIlwain Tissue Chopper</td>
			<td style="width:183px;">Ted Pella</td>
			<td style="width:151px;">10180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Melanoma gp100 antibody</td>
			<td style="width:183px;">Abcam</td>
			<td style="width:151px;">ab137078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Nutrient Mix F-12 Ham&#39;s Media</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">N6760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phorbol 12-Myristate 13-acetate</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Pierce 16% Formaldehyde</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:151px;">28908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Porcine Trypsin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">85450C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">RPMI 1640 media</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Saponin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">S-7900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tissue Chopper Blade</td>
			<td style="width:183px;">Ted Pella</td>
			<td style="width:151px;">121-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tissue Chopper Plastic Disk</td>
			<td style="width:183px;">Ted Pella</td>
			<td style="width:151px;">10180-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Trypsin</td>
			<td style="width:183px;">VWR</td>
			<td style="width:151px;">VWRL0154-0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid generation of primary murine melanocyte and fibroblast cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

Male and female C57Bl/6J mice were euthanized on postnatal days 0-4 and the truncal skin was subjected to mechanical dissociation as described above. After chopping, the skin formed a viscous slurry lacking any sign of structural tissue. Centrifugation of this slurry resulted in the formation of a large cell pellet at the bottom of the conical tube and a layer of adipose floating on top of the supernatant. This adipose layer was discarded with the supernatant while the remaining cell pellet was resuspended and transferre...

Watch this Scientific Journal Video about Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures at JoVE.com

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Generazione rapida delle culture primarie di Murine Melanocite e Fibroblast

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

rapid-generation-of-primary-murine-melanocyte-and-fibroblast-cultures

Research

JoVE Journal

Cancer Research

10.6K Views.  The Ohio State University. Questo protocollo descrive un metodo rapido e semplice per generare colture primarie di melanociti e fibroblasti dai topi. Poiché questa tecnica non richiede che l'epidermide e il derma siano separati, può essere eseguita in modo rapido e coerente con un allenamento minimo. Questo metodo può essere utilizzato per studiare la biologia dei melanociti cutanei e dei fibroblasti.Gli esperimenti in queste culture possono fornire informazioni rilevanti per il cancro, la guarigione delle fe...

Video: Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Un modello murino di fatica indotta da irradiazione Peripheral

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Ricerca

Ricerca sul cancro

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living biobanks&#34; of patient derived cell lines. For many types of epithelial cells, various three dimensional (3D) culture approaches have been described that support an improved differentiated state. While CRCs retain their lineage commitment to the tissue from which they are isolated, they fail to express many of the differentiation markers associated with the tissue of origin when grown under normal two dimensional (2D) culture conditions. To enhance the application of patient-derived CRCs for prostate cancer research, a 3D culture format has been defined that enables a rapid (2 weeks total) luminal cell differentiation in both normal and tumor-derived prostate epithelial cells. Herein, a filter insert-based format is described for the culturing and differentiation of both normal and malignant prostate CRCs. A detailed description of the procedures required for cell collection and processing for immunohistochemical and immunofluorescent staining are provided. Collectively the 3D culture format described, combined with the primary CRC lines, provides an important medium- to high- throughput model system for biospecimen-based prostate research.

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

Un sistema di coltura 3D Rapid Cartuccia filtro-based per la prostata primaria cellulare Differenziazione

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Immunophenotyping di Orthotopic omotrapianto (Syngeneic) dell'Adenocarcinoma Ductal pancreatico murino primario KPC tramite flusso Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Controllo spaziale e temporale dell'iniziazione del melanoma murino dalle cellule staminali di melanociti mutanti

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

Sistema a matrice 3D derivato da fibroblasti applicabile al saggio di formazione di tubi endoteliali

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Un nuovo modello di spheroid del tumore strutturato in 3D modulato come fibromastro per studiare l'interazione tumora-stroma e la scoperta di farmaci

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Generazione di xenografti larvali zebrafish e analisi del comportamento tumorale

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Isolamento di fibroblasti primari associati al cancro da un modello murino singeneico di cancro al seno per lo studio di nanoparticelle mirate

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Profilazione spaziale dell'espressione di proteine e RNA nei tessuti: un approccio alla messa a punto della microdissezione virtuale

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

Generazione ed espansione delle linee tumorali primarie e maligne del mesotelioma pleurico

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...