Name: murine dermal fibroblast isolation by facs
Uploaded: 2016-01-07T10:00:01.000+00:00
Duration: 6 min 4 s
Description: Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

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 mm.</li>
	<li>Submerge the mouse in 70% ethanol and place on a clean, sterile surface to dry.</li>
	<li>Immediately harvest dorsal mouse skin using sterile dissecting scissors. In female mice, avoid including the mammary tissue.</li>
	<li>Starting the base of the tail, use forceps to tent up the skin and make a transverse cut before dissecting along the supra-fascial plane.</li>
	<li>Carefully avoid including any subcutaneous fat while harvesting the skin. Examine the harvested skin for any subcutaneous fat and carefully scrape it off using the blunt edge of a scalpel.</li>
	<li>Rinse the harvested skin in betadine followed by 5x PBS washes on ice. 
	Note: It is important to keep the skin as close to sterile as possible to avoid contamination.</li>
	<li>Mince the skin using razor blades and dissecting scissors in a sterile dish until the sample is of a uniform consistency with 2-3 mm pieces.</li>
	<li>Prepare 50 ml conical tubes containing 20 ml collagenase IV at a concentration of 1 mg/ml in DMEM. Divide the dermis into the tubes on the basis of five mice per tube.</li>
	<li>Agitate samples vigorously while incubating at 37 &#176;C for 1 hr in either a water bath or oven.</li>
	<li>Remove samples from the incubator and pass through a 10 ml syringe without a needle 3-5x in a sterile hood.</li>
	<li>Place the samples back into the incubator at 37 &#176;C and shake vigorously for a further 30 min.</li>
	<li>In a sterile hood, pipette the samples up and down 3-5x using a 10 ml pipette. Pipette the sample through a 100 &#181;m filter into a new 50 ml conical tube.</li>
	<li>Pass 20 ml of 10% FBS DMEM through the same filter to maximize cell yield and bring the total volume to 40 ml. Centrifuge at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove the supernatant using a sterile glass pipette, taking great care to first remove the upper fat layer prior to remaining supernatant. 
	Note: This step is critical to reduce adipocyte contamination.</li>
	<li>Resuspend the pellets in 20 ml 10% FBS DMEM.</li>
	<li>Pass the cell/DMEM suspension through a 70 &#181;m filter.</li>
	<li>Rinse the filter with 10 ml 10% FBS DMEM and centrifuge the filtered suspension at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove the supernatant using a sterile glass pipette, again taking care to first remove any remaining fat layer.</li>
	<li>If there is significant RBC contamination (the pellet is visibly red), re-suspend the pellets in 20 ml ACK lysis buffer and incubate for 5 min at RT. Otherwise skip to step 24.</li>
	<li>Add an equal volume (20 ml) of FACS buffer (PBS, 10% FBS, 0.1% sodium azide), then mix, and keep aside a 5 ml aliquot as an unstained control. Centrifuge the remaining sample at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove supernatant and put pellet on ice. The cells may be frozen down at this point if FACS time is not available.</li>
</ol>
2. Isolation of Fibroblasts by FACS
<ol>
	<li>Make 500 &#181;l of lineage antibody incubation mix for each pellet. Do this by first adding 475 &#181;l of FACS buffer containing DNase (10 &#181;g/ml) to a tube, and then adding fluorophore-conjugated CD31 (1:100), CD45 (1:200), Tie2 (1:50), Ter-119 (1:200), and EpCAM (1:100) antibodies to achieve the respective dilution for each antibody.</li>
	<li>Re-suspend each pellet in 500 &#181;l of lineage antibody incubation mix and incubate this suspension on ice for 20 min.</li>
	<li>Add 5 ml FACS buffer containing DNase (10 &#181;g/ml) to the sample and gently mix. Centrifuge at 300 g for 8 min at 4 &#176;C.</li>
	<li>Remove the supernatant and wash the cell pellet with 5ml FACS buffer containing DNase (10 &#181;g/ml) and centrifuge using the same conditions as in step 26.</li>
	<li>Resuspend the pellet in 500 &#181;l FACS buffer containing DNase (10 &#181;g/ml) and put aside a 50 &#181;l aliquot as a viability dye control.</li>
	<li>Add viability dye of choice to the remaining sample in the concentration indicated for the chosen dye.</li>
	<li>Perform FACS analysis31 and sorting for Viability Dye-/CD31-/CD45-/Tie2-/Ter-119-/EpCAM- cells (see Figure 2A). Sort directly into FACS buffer.</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr><tr><td>Collagenase IV</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>Elastase</td><td>Abcam</td><td>ab95133</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>A14430-01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) lysing buffer</td><td>Gibco</td><td>A10492-01</td><td></td></tr><tr><td>40 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>100 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>CD31</td><td>BioLegend</td><td>102421</td><td></td></tr><tr><td>CD45</td><td>BioLegend</td><td>103125</td><td></td></tr><tr><td>Tie2</td><td>BioLegend</td><td>124005</td><td></td></tr><tr><td>Ter-119</td><td>BioLegend</td><td>116233</td><td></td></tr><tr><td>EpCAM (CD326)</td><td>eBioscience</td><td>48-5791</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>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 ...

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Research

JoVE Journal

Developmental Biology

20.9K Views.  Stanford University School of Medicine. 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.

Video: Murine Dermal Fibroblast Isolation by FACS

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

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.

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 ...

Cancer Research

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetics

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioengineering

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Biology

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Medicine

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Rapid Culturing of Primary Murine Melanocytes and Fibroblasts: Isolating Melanocytes and Fibroblasts from Whole Murine Skin Sample

Source: Murphy, B. M. et al. <a href="https://www.jove.com/v/59468"> Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures</a>. J. Vis. Exp. (2019)
This video describes the rapid method to isolate and culture the melanocytes and fibroblast simultaneously from the postnatal mouse pup&#8217;s skin. These cells can be maintained and manipulated in vitro to study skin cell biology, wound healing, and melanoma.

Source: Murphy, B. M. et al.  Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures. J. Vis. Exp. (2019)
This video describes the rapid ...