This work was supported in part by a grant from NIH grant R01 GM087609 (to H.P.L.), a Gift from Ingrid Lai and Bill Shu in honor of Anthony Shu (to H.P.L.), NIH grant U01 HL099776 (to M.T.L.), the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation (to M.T.L., G.C.G. and H.P.L.). G.G.W. was supported by the Stanford School of Medicine, the Stanford Medical Scientist Training Program, and NIGMS training grant GM07365. Z.N.M. was supported by the Plastic Surgery Foundation Research Fellowship Grant and the Hagey Family Fund. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159, the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award, and the Transplant and Tissue Engineering Fellowship Award.

This protocol follows methods approved by the Stanford University Administrative Panel on Laboratory Animal Care.
1. Digestion of Murine Dermis
<ol>
	<li>Euthanize mice by cervical dislocation after anesthesia with an intraperitoneal injection of ketamine 100 mg/kg + xylazine 20 mg/kg + acepromazine 3 mg/kg. 
	Note: Various ages and backgrounds can be used.</li>
	<li>Shave and depilate the dorsal skin. Approximately 100,000 cells can be isolated from a piece of dorsal skin 60 mm x 100 m...

<ol>
	<li>Sorrell, J. M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts-a+diverse+population+at+the+center+of+it+all.">Fibroblasts-a diverse population at the center of it all.</a> International review of cell and molecular biology. 276, 161-214 (2009).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+fibrosis.">Cellular and molecular mechanisms of fibrosis.</a> The Journal of pathology. 214, 199-210 (2008).</li><li>Powell, D. W., 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=Myofibroblasts.+I.+Paracrine+cells+important+in+health+and+disease.">Myofibroblasts. I. Paracrine cells important in health and disease.</a> The American journal of physiology. 277, C1-C9 (1999).</li><li>Wilson, M. S., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+fibrosis:+pathogenesis,+etiology+and+regulation.">Pulmonary fibrosis: pathogenesis, etiology and regulation.</a> Mucosal immunology. 2, 103-121 (2009).</li><li>Hinz, B., 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=The+myofibroblast:+one+function,+multiple+origins.">The myofibroblast: one function, multiple origins.</a> The American journal of pathology. 170, 1807-1816 (2007).</li><li>Li, B., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+and+myofibroblasts+in+wound+healing:+force+generation+and+measurement.">Fibroblasts and myofibroblasts in wound healing: force generation and measurement.</a> J Tissue Viability. 20, 108-120 (2011).</li><li>Wong, J. W., 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=Wound+healing+in+oral+mucosa+results+in+reduced+scar+formation+as+compared+with+skin:+evidence+from+the+red+Duroc+pig+model+and+humans.">Wound healing in oral mucosa results in reduced scar formation as compared with skin: evidence from the red Duroc pig model and humans.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 17, 717-729 (2009).</li><li>Szpaderska, A. M., Zuckerman, J. D., DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+injury+responses+in+oral+mucosal+and+cutaneous+wounds.">Differential injury responses in oral mucosal and cutaneous wounds.</a> Journal of dental research. 82, 621-626 (2003).</li><li>Hantash, B. M., Zhao, L., Knowles, J. A., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+and+fetal+wound+healing.">Adult and fetal wound healing.</a> Frontiers in bioscience : a journal and virtual library. 13, 51-61 (2008).</li><li>Buchanan, E. P., Longaker, M. T., Lorenz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+skin+wound+healing.">Fetal skin wound healing.</a> Advances in clinical chemistry. 48, 137-161 (2009).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors:+wounds+that+do+not+heal.+Similarities+between+tumor+stroma+generation+and+wound+healing.">Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing.</a> The New England journal of medicine. 315, 1650-1659 (1986).</li><li>Dumont, N., 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=Breast+fibroblasts+modulate+early+dissemination,+tumorigenesis,+and+metastasis+through+alteration+of+extracellular+matrix+characteristics.">Breast fibroblasts modulate early dissemination, tumorigenesis, and metastasis through alteration of extracellular matrix characteristics.</a> Neoplasia. 15, 249-262 (2013).</li><li>Servais, C., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+sentinel+cells+to+inflammatory+culprits:+cancer-associated+fibroblasts+in+tumour-related+inflammation.">From sentinel cells to inflammatory culprits: cancer-associated fibroblasts in tumour-related inflammation.</a> The Journal of pathology. 229, 198-207 (2013).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer:+a+novel+tumor-promoting+cell+type.">Stromal fibroblasts in cancer: a novel tumor-promoting cell type.</a> Cell cycle. 5, 1597-1601 (2006).</li><li>Li, X., 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=Targeting+the+cancer-stroma+interaction:+a+potential+approach+for+pancreatic+cancer+treatment.">Targeting the cancer-stroma interaction: a potential approach for pancreatic cancer treatment.</a> Current pharmaceutical design. 18, 2404-2415 (2012).</li><li>Sorrell, J. M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+heterogeneity:+more+than+skin+deep.">Fibroblast heterogeneity: more than skin deep.</a> Journal of cell science. 117, 667-675 (2004).</li><li>Cormack, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ham's Histology. , 450-474 (1987).</li><li>Jahoda, C. A., Reynolds, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal-epidermal+interactions.+Adult+follicle-derived+cell+populations+and+hair+growth.">Dermal-epidermal interactions. Adult follicle-derived cell populations and hair growth.</a> Dermatologic clinics. 14, 573-583 (1996).</li><li>Jahoda, C. A., Reynolds, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+dermal+sheath+cells:+unsung+participants+in+wound+healing.">Hair follicle dermal sheath cells: unsung participants in wound healing.</a> Lancet. 358, 1445-1448 (2001).</li><li>Sorrell, J. M., Baber, M. A., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+bilayered+dermal+equivalent+containing+human+papillary+and+reticular+dermal+fibroblasts:+use+of+fluorescent+vital+dyes.">Construction of a bilayered dermal equivalent containing human papillary and reticular dermal fibroblasts: use of fluorescent vital dyes.</a> Tissue engineering. 2, 39-49 (1996).</li><li>Harper, R. A., Grove, G. <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+fibroblasts+derived+from+papillary+and+reticular+dermis:+differences+in+growth+potential+in+vitro.">Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro.</a> Science. 204, 526-527 (1979).</li><li>Schafer, I. A., Pandy, M., Ferguson, R., Davis, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+observation+of+fibroblasts+derived+from+the+papillary+and+reticular+dermis+of+infants+and+adults:+growth+kinetics,+packing+density+at+confluence+and+surface+morphology.">Comparative observation of fibroblasts derived from the papillary and reticular dermis of infants and adults: growth kinetics, packing density at confluence and surface morphology.</a> Mechanisms of ageing and development. 31, 275-293 (1985).</li><li>Sorrell, J. M., Baber, M. A., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-matched+papillary+and+reticular+human+dermal+fibroblasts+differ+in+their+release+of+specific+growth+factors/cytokines+and+in+their+interaction+with+keratinocytes.">Site-matched papillary and reticular human dermal fibroblasts differ in their release of specific growth factors/cytokines and in their interaction with keratinocytes.</a> Journal of cellular physiology. 200, 134-145 (2004).</li><li>Sharon, Y., Alon, L., Glanz, S., Servais, C., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+normal+and+cancer-associated+fibroblasts+from+fresh+tissues+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS).</a> Journal of visualized experiments : JoVE. , e4425 (2013).</li><li>Walmsley, G. 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The protocol described in this manuscript offers a means to isolate fibroblasts by FACS-based sorting, in comparison to existing methods, which either select for a subpopulation or require time in cell culture before subsequent analyses. The time required from harvesting of the skin to sorting of fibroblasts is approximately 6 hr; however, the number of mice used in the harvest will influence this estimate.
Several points in the protocol require particular care. The first is limiting adipocyte...

Fibroblasts are frequently defined morphologically as spindle-shaped cells that adhere to plastic substrates. Fibroblasts are the principle cell type responsible for synthesizing and remodeling the extracellular matrix in embryonic and adult organs1. Fibroblasts are thus critical to mammalian development and contribute substantially to the extracellular milieu that influences the behavior of neighboring cell types present in each tissue and organ.
Fibroblasts are also the principal cell type behind a diverse set of medical conditions that cause enormous clinical burden. Pathologic fibroblast activity impairs normal tissue function and includes tissue and organ fibrosis (such as the lung and liver), scarring following cutaneous wound healing, atherosclerosis, systemic sclerosis, and formation of atheromatous plaques after blood vessel injury2-5. Wound healing in particular, both acutely and chronically, involves deposition of scar tissue that neither resembles nor functions like the normal tissue surrounding it, and leads to significant morbidity across diverse pathologic states. Following injury, there is a transition of fibroblasts to myofibroblasts, which then secrete structural ECM components, exert paracrine effects on neighboring cell types, and restore mechanical stability by depositing scar tissue6.
In cutaneous tissues there exists significant variation in the quality of wound repair across developmental time and between anatomic sites. In the first two trimesters of life the fetus heals without scarring; however, from the third trimester on and throughout adulthood, humans heal with a scar. Site-specific, in addition to age-specific, differences in wound healing exist. Wounds in the oral cavity remodel with minimal scar formation7,8, while scar tissue deposition within cutaneous wounds is significant9. Controversy persists concerning the relative influence of the environment versus the intrinsic properties of local fibroblasts on the outcome of wound healing in regards to both age and location10,11. Given the significant differences in the healing of mouse oral vs. cutaneous dermis and earlier embryonic (E15) vs. later embryonic (E18) dermis, it is likely that intrinsic differences in the populations of fibroblasts at certain developmental ages and among various anatomic sites exist.
In 1986, Harold F. Dvorak posited tumors are wounds that do not heal12. Dvorak concluded that tumors behave like wounds in the body and induce their stroma by activating the wound healing response of the host. Numerous studies have since investigated the contribution of fibroblasts to the progression of carcinomas13-15, but as in the case of wound healing, the identity and embryonic origin of the fibroblasts that contribute to the stromal compartment of cutaneous carcinomas has not been adequately defined. The answer to this question bears medical relevance given recent studies exposing the tumor-associated fibroblast as a potentially effective target for anti-cancer therapy16.
Identifying and prospectively isolating the fibroblast lineages endowed with fibrogenic potential in vivo is an essential step towards effectively manipulating their response to injury across a wide range of acute and chronic disease states. In 1987, Cormack demonstrated two subpopulations of fibroblasts, one residing within the papillary and one within the reticular dermis17,18. A third subpopulation was found associated with hair follicles in the dermal papilla region of the follicle19,20. When cultured, these fibroblast subtypes exhibit differences in growth potential, morphology, and growth factor/cytokines profiles21-24.
To date, studies examining fibroblast heterogeneity have largely failed to adequately characterize developmental and functional diversity among fibroblasts in vivo. This is, in part, is a result of a reliance on cultured fibroblast populations and the homogenizing effect of cell culture or positive selection on the basis of a self surface receptor not expressed by all fibroblasts25. A recent study from our lab demonstrated a profound surface marker and transcriptional shift in cultured vs. uncultured fibroblasts isolated by the FACS-based isolation methodology presented in this manuscript26.
Subsequently, we identified a specific fibroblast lineage within the murine dorsal dermis and determined that this lineage, defined by embryonic expression of Engrailed-1, is primarily responsible for connective tissue deposition in the dorsal skin. The lineage functions during both acute and chronic forms of fibrosis including wound healing, cancer stroma formation, and radiation induced fibrosis27. The characterization of distinct fibroblast lineages has critical implications for therapies aimed at modulating fibrogenic behavior.
Rather than using existing protocols that rely upon in vitro manipulation to achieve cell isolation28,29, the harvest protocol (Figure 1) detailed here will help yield informative analyses of fibroblasts that more accurately capture phenotype and behavior in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="1000">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">Surgical Forceps</td>
			<td style="width:369px;">Kent Scientific</td>
			<td style="width:108px;">INS650916</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elastase</td>
			<td>Abcam</td>
			<td>ab95133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>A14430-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium-Chloride-Potassium (ACK) lysing buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 micron filters</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD31</td>
			<td>BioLegend</td>
			<td>102421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD45</td>
			<td>BioLegend</td>
			<td>103125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tie2</td>
			<td>BioLegend</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ter-119</td>
			<td>BioLegend</td>
			<td>116233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EpCAM (CD326)</td>
			<td>eBioscience</td>
			<td>48-5791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">propidium iodide (PI viability stain)</td>
			<td>BioLegend</td>
			<td>421301</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine dermal fibroblast isolation by facs

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

The validity of this approach (Figure 1) has been verified in a number of ways, which can be examined in detail in our recent publication27. These include immunocytochemistry of sorted cells and mass cell and single cell transcriptional analysis of freshly sorted cells. Sorting fibroblasts directly rather than relying on culture more accurately captures their in vivo phenotype. Using a lineage negative depletion approach (Figure 2A) rather than a positive selection approach avoids pre-sel...

Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

Murine Dermal Fibroblast Isolation by FACS

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

The overall goal of this procedure is to isolate fibroblasts using FACS, thereby foregoing in vitro culture, which has been shown to cause a phenotypic change in these cells. This method can help answer key questions in developmental biology and wound healing, such as how to identify fibroblast subtypes involved in scarring and fibrosis. The main advantage of this technique is that in vitro experiments are avoided as adherence to plastic, male to the phenotype of the fibroblast.Not only can this method provide insights into dermal fibroblasts, but it can also be applied to other systems, such as peritoneal fibroblasts and abdominal adhesions. Begin by shaving and depilating the dorsal skin of the mouse of interest. Then, submerge the denuded mouse in 70%ethanol and place the animal on a clean, sterile surface to dry.Next, starting at the base of the tail, use forceps to tent the skin and make a transverse cut with dissecting scissors. Then, dissect along the suprafascial plane to harvest the dorsal tissue. When a 60 by 100 millimeter piece of skin has been removed, use the blunt edge of a scalpel to carefully remove any subcutaneous fat.Then, rinse the harvested skin in betadine, followed by five PBS washes on ice. Using razorblades and dissecting scissors, mince the pieces of dermis in a sterile dish until the samples are uniformly approximately two to three millimeters in size. Then, transfer tissue fragments from up to five mice into the appropriate number of individual 50 milliliter conical tubes containing 20 milliliters of collagenase 4 in dmem.Agitate the samples vigorously at 37 degrees Celsius. After one hour, transfer the tubes to a sterile hood and pass the tissue flurries through a needless 10 milliliter syringe three to five times. Return the samples to the shaker for another 30 minutes, followed by another three to five pipettes with a 10 milliliter syringe.Then, filter the samples through a 100 micron strainer into a new 50 milliliter tube and wash the mesh with 20 milliliters of dmem, supplemented with FBS to bring the total volume up to 40 milliliters. Next, spin down the cells and use a sterile glass pipette to aspirate the upper fat layer. When the contaminating adipose sites have been removed, use a new glass pipette to remove the rest of the supernatant and resuspend the pellets in 20 milliliters of dmem plus FBS.Now, filter the cells through a 75 micron strainer and rinse the mesh with 10 milliliters of dmem plus FBS. Then, spin down the cells again and remove the fat and supernatant as just demonstrated. Resuspend the pellet in 20 milliliters of FACS buffer, setting aside a five milliliter aliquot for the unstained control.Then, spin down the remaining sample, discard the supernatant and place the pellet on ice. To isolate the fibroblasts by FACS, resuspend the pellets in 500 microliters of lineage antibody incubation mix on ice. After 20 minutes, gently mix five milliliters of FACS buffer, supplemented with DNase with the cells and spin them down.Wash the pellet in a second five milliliters of DNase. Then, resuspend the cells in 500 microliters of FACS buffer and DNase, reserving a 50 microliter aliquot of cells for the viability dye control. Finally, add viability dye to the remaining sample and sort the cells according to the diagram.Using a lineage negative depletion approach rather than a positive selection approach avoids pre-selecting for particular subpopulations. Pull transcriptome microarray analysis reveals that cultured fibroblasts isolated by the live harvest and tissue explant methodologies have a high degree of similarity at a transcriptome wide level with a Pearson product-moment correlation coefficient of 0.92. By comparison, cultured fibroblasts differed significantly from a live harvested uncultured fibroblast, establishing the importance of analyzing live harvested fibroblasts over cultured fibroblasts.After it's development, this technique paved the way for researchers in the field of developmental biology and wound healing to confirm fibroblasts heterogeneity and identify subpopulations responsible for scarring and other forms of fibroses.

murine-dermal-fibroblast-isolation-by-facs

Research

JoVE Journal

Developmental Biology

20.5K vistas.  Stanford University School of Medicine. The overall goal of this procedure is to isolate fibroblasts using FACS, thereby foregoing in vitro culture, which has been shown to cause a phenotypic change in these cells. This method can help answer key questions in developmental biology and wound healing, such as how to identify fibroblast subtypes involved in scarring and fibrosis. The main advantage of this technique is that in vitro experiments are avoided as adherence to plastic, male to the phenotype of the fibroblast.Not onl...