Name: isolation and characterization of adult cardiac fibroblasts and myofibroblasts
Uploaded: 2020-03-12T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts at JoVE.com

The authors want to thank Dr. Ivo Kalajzic for the &#945;SMA-GFP mice. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award Number R01GM118300 (S.S.), National Institute of Biomedical Imaging and Bioengineering of the NIH under Award Number R21EB019509 (P.P.Y.), and Scientist Development Grant of the American Heart Association under Award Number 17SDG33630187 (S.S.). Flow cytometry analyses were performed at the VUMC Flow Cytometry Shared Resource which is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404).

This study strictly upholds the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Vanderbilt University Institutional Animal Care and Use Committee approved the protocol (protocol number: M1600076-01).
1. Heart Dissection
<ol>
	<li>Solution preparation
	<ol>
		<li>KHB buffer
		<ol>
			<li>Using a stir bar, slowly dissolve 9.4 g of Krebs-Henseleit buffer (KHB) powder in 900 mL of DDI water. 
			NOTE: Buffer ...

<ol>
	<li>Ivey, M. J., Tallquist, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+Cardiac+Fibroblast.">Defining the Cardiac Fibroblast.</a> Circulation Journal. 80 (11), 2269-2276 (2016).</li><li>Prabhu, S. D., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biological+Basis+for+Cardiac+Repair+After+Myocardial+Infarction:+From+Inflammation+to+Fibrosis.">The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis.</a> Circulatory Research. 119 (1), 91-112 (2016).</li><li>Duan, 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=Wnt1/betacatenin+injury+response+activates+the+epicardium+and+cardiac+fibroblasts+to+promote+cardiac+repair.">Wnt1/betacatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair.</a> EMBO Journal. 31 (2), 429-442 (2012).</li><li>Shinde, A. V., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+myocardial+infarction:+a+role+in+inflammation+and+repair.">Fibroblasts in myocardial infarction: a role in inflammation and repair.</a> Journal of Molecular and Cellular Cardiology. 70, 74-82 (2014).</li><li>Saraswati, S., Marrow, S. M. W., Watch, L. A., Young, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+pro-angiogenic+functional+role+for+FSP1-positive+fibroblast+subtype+in+wound+healing.">Identification of a pro-angiogenic functional role for FSP1-positive fibroblast subtype in wound healing.</a> Nature Communications. 10 (1), 3027 (2019).</li><li>Kong, P., Christia, P., Saxena, A., Su, Y., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+specificity+of+fibroblast-specific+protein+1+in+cardiac+remodeling+and+fibrosis.">Lack of specificity of fibroblast-specific protein 1 in cardiac remodeling and fibrosis.</a> American Journal of Physiology -Heart and Circulatory Physiology. 305 (9), 1363-1372 (2013).</li><li>Furtado, M. B., Nim, H. T., Boyd, S. E., Rosenthal, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=View+from+the+heart:+cardiac+fibroblasts+in+development,+scarring+and+regeneration.">View from the heart: cardiac fibroblasts in development, scarring and regeneration.</a> Development. 143 (3), 387-397 (2016).</li><li>Stellato, M., Czepiel, M., Distler, O., Blyszczuk, P., Kania, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Isolation+of+Cardiac+Fibroblasts+From+the+Adult+Mouse+Heart+Using+Two-Color+Flow+Cytometry.">Identification and Isolation of Cardiac Fibroblasts From the Adult Mouse Heart Using Two-Color Flow Cytometry.</a> Frontiers in Cardiovascular Medicine. 6, 105 (2019).</li><li>Ackers-Johnson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified,+Langendorff-Free+Method+for+Concomitant+Isolation+of+Viable+Cardiac+Myocytes+and+Nonmyocytes+From+the+Adult+Mouse+Heart.">A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart.</a> Circulatory Research. 119 (8), 909-920 (2016).</li><li>Saraswati, S., Marrow, S. M. W., Watch, L. A., Young, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+pro-angiogenic+functional+role+for+FSP1-positive+fibroblast+subtype+in+wound+healing.">Identification of a pro-angiogenic functional role for FSP1-positive fibroblast subtype in wound healing.</a> Nature Communications. 10 (1), 3027 (2019).</li><li>Kalajzic, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+alpha-smooth+muscle+actin+GFP+reporter+to+identify+an+osteoprogenitor+population.">Use of an alpha-smooth muscle actin GFP reporter to identify an osteoprogenitor population.</a> Bone. 43 (3), 501-510 (2008).</li><li>Travers, J. G., Kamal, F. A., Robbins, J., Yutzey, K. E., Blaxall, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Fibrosis:+The+Fibroblast+Awakens.">Cardiac Fibrosis: The Fibroblast Awakens.</a> Circulatory Research. 118 (6), 1021-1040 (2016).</li><li>Bell, E., Ivarsson, B., Merrill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+tissue-like+structure+by+contraction+of+collagen+lattices+by+human+fibroblasts+of+different+proliferative+potential+in+vitro.">Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.</a> Proceedings of the National Academy of Sciences USA. 76 (3), 1274-1278 (1979).</li><li>Montesano, R., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta+stimulates+collagen-matrix+contraction+by+fibroblasts:+implications+for+wound+healing.">Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing.</a> Proceedings of the National Academy of Sciences USA. 85 (13), 4894-4897 (1988).</li><li>Goldsmith, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+fibroblasts+in+the+heart.">Organization of fibroblasts in the heart.</a> Developmental Dynamics. 230 (4), 787-794 (2004).</li><li>Bagchi, R. A., Lin, J., Wang, R., Czubryt, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fibronectin+gene+expression+in+cardiac+fibroblasts+by+scleraxis.">Regulation of fibronectin gene expression in cardiac fibroblasts by scleraxis.</a> Cell Tissue Research. 366 (2), 381-391 (2016).</li><li>Goodpaster, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunohistochemical+method+for+identifying+fibroblasts+in+formalin-fixed,+paraffin-embedded+tissue.">An immunohistochemical method for identifying fibroblasts in formalin-fixed, paraffin-embedded tissue.</a> Journal of Histochemistry and Cytochemistry. 56 (4), 347-358 (2008).</li><li>Chapman, D., Weber, K. T., Eghbali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fibrillar+collagen+types+I+and+III+and+basement+membrane+type+IV+collagen+gene+expression+in+pressure+overloaded+rat+myocardium.">Regulation of fibrillar collagen types I and III and basement membrane type IV collagen gene expression in pressure overloaded rat myocardium.</a> Circulatory Research. 67 (4), 787-794 (1990).</li><li>Vasquez, C., Benamer, N., Morley, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cardiac+fibroblast:+functional+and+electrophysiological+considerations+in+healthy+and+diseased+hearts.">The cardiac fibroblast: functional and electrophysiological considerations in healthy and diseased hearts.</a> Journal of Cardiovascular Pharmacology. 57 (4), 380-388 (2011).</li><li>Hudon-David, F., Bouzeghrane, F., Couture, P., Thibault, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thy-1+expression+by+cardiac+fibroblasts:+lack+of+association+with+myofibroblast+contractile+markers.">Thy-1 expression by cardiac fibroblasts: lack of association with myofibroblast contractile markers.</a> Journal of Molecular and Cellular Cardiology. 42 (5), 991-1000 (2007).</li><li>Kanisicak, O., 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=Genetic+lineage+tracing+defines+myofibroblast+origin+and+function+in+the+injured+heart.">Genetic lineage tracing defines myofibroblast origin and function in the injured heart.</a> Nature Communications. 7, 12260 (2016).</li><li>Pinto, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+Cardiac+Cellular+Composition.">Revisiting Cardiac Cellular Composition.</a> Circulatory Research. 118 (3), 400-409 (2016).</li></ol>

Fibroblasts are a heterogenous group of cells, identified by diverse set of markers. The protein markers that have been used to identify fibroblasts are discoidin domain receptor 2 (DDR2), fibronectin, vimentin, collagen I and III, and Thy115,16,17,18,19,20. Whereas vimentin has been used to identify uninjured quiescent cardi...

Cardiac fibroblasts, cells of mesenchymal origin, play a significant role in maintaining the electrical conduction and mechanical forces in the heart in addition to the maintenance of cardiac architecture during homeostasis1. Following injury, these cells are activated, expand, and produce extracellular matrix (ECM) proteins2. Many preclinical studies have revealed fibroblasts as critical cellular regulators that maintain the structural integrity of an injured heart3 as well as main effector cells responsible for unchecked production and deposition of ECM proteins, resulting in stiff scar formation and heart failure4. Fibroblasts are a heterogenous group of cells, making it challenging to dissect their reparative function from pro-fibrotic maladaptive properties. Recently, the functional heterogeneity of two distinct fibroblast subtypes following myocardial injury have been defined, indicating the possibility of isolating different fibroblast subtypes and studying their role in wound healing5.
Obtaining a pure fibroblast population is crucial in delineating their functional role in repair and fibrosis. However, the presence of multiple fibroblast markers that recognize other cell types make it challenging to isolate a substantially pure fibroblast population6. Several elegant studies have devised clever ways to isolate cardiac fibroblasts from uninjured and injured myocardium. The most popular and well-established method of enriching fibroblasts is through selective adhesion following enzymatic tissue digestion7.
Additionally, fluorescence-activated cell sorting (FACS) of fibroblasts based on cell surface antigens has been successfully described8. In the study, following enzymatic digestion, the mesenchymal cells were sorted as lineage-negative (Lin: Ter119&#8722;CD45&#8722;CD31&#8722;) and gp38-positive (gp38+) from mouse hearts. Gp38+ve cells were confirmed to be fibroblasts based on their co-expression of col1&#945;1 and other mesenchymal markers. Although most tissue digestion is completed after dissecting out the ventricle in a Petri dish, a recent study has investigated the use of a direct needle enzyme perfusion of the left ventricle to isolate myocytes and non-myocytes which include fibroblasts9. Fibroblasts were then isolated by selective adhesion in this case.
This protocol describes the isolation and enrichment of fibroblasts using three methods. The first is an already established method involving selective adhesion of fibroblasts following enzymatic digestion. The second method is used to primarily isolate injury-induced alpha smooth muscle expressing myofibroblasts. The third method involves sequential, magnetic depletion of an enzyme-digested cardiac cell suspension of hematopoietic and endothelial cells. Following depletion, fibroblasts/myofibroblasts are isolated based on the presence of the antigen MEFSK4 using magnetic beads. Recently, MEFSK4 has been described as an antigen present on quiescent as well as activated fibroblasts, making it a suitable marker for fibroblast identification and isolation. Naturally, all the methods described here have unique limitations. It is therefore highly recommended to check the purity of the isolated cell population by flow analysis, immunostaining, and semi-quantitative real-time PCR. However, these methodologies can be expanded upon, and additional markers can be added in order to exclude other contaminating populations prior to utilizing the fibroblast and myofibroblast populations for crucial experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fungal (Amphotericin B-solubilized; Fungizone)</td>
			<td>Sigma Aldrich</td>
			<td>A9528</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serium Albumin (BSA)</td>
			<td>Sigma</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase blend (Liberase Blendzyme 3 TH)</td>
			<td>Roche Applied Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DDI water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DI water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 with L-Glutamine and HEPES</td>
			<td>Life technologies</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I(20U/mL)</td>
			<td>BioRad</td>
			<td>7326828</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline (dPBS) without Ca2+ and Mg2+</td>
			<td>Gibco</td>
			<td>13190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FC Blocker (Purified anti-mouse CD16/CD32)</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life technologies</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>10% goat serum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS) with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>21-023-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>Corning</td>
			<td>25-060-Ci</td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Henseleit Buffer powder</td>
			<td>Sigma</td>
			<td>K3753</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycoplasma prophylactic (Plasmocin)</td>
			<td>Invivogen</td>
			<td>ant-mpp</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Red Blood Cell Lysis Buffer</td>
			<td>Miltenyi</td>
			<td>130-094-183</td>
			<td></td>
		</tr>
		<tr>
			<td>Slow-fade Mounting Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 Rat Collagen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7AAD (stock: 1 mg/mL solution in DMSO)</td>
			<td>Molecular Probes</td>
			<td>A1310</td>
			<td>dilution = 1:1000; RRID =</td>
		</tr>
		<tr>
			<td>CD45-APC</td>
			<td>BD Bioscience</td>
			<td>559864</td>
			<td>dilution = 1:200; RRID = AB_398672</td>
		</tr>
		<tr>
			<td>CD31-PE</td>
			<td>BD Bioscience</td>
			<td>553373</td>
			<td>dilution = 1:200; RRID = AB_394819</td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>BD Biosciences</td>
			<td>553370</td>
			<td>dilution = 1:250; RRID = AB_394816</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>BD Biosciences</td>
			<td>553076</td>
			<td>dilution = 1:250; RRID = AB_394606</td>
		</tr>
		<tr>
			<td>COL 1&#945;1</td>
			<td>MD Bioproducts</td>
			<td>203002</td>
			<td>dilution = 1:1000; RRID =</td>
		</tr>
		<tr>
			<td>Ghost dye violet 510 (Formulation: 1 uL/test in DMSO)</td>
			<td>Tonbo Biosciences</td>
			<td>13-0870</td>
			<td>dilution = 1:1000; RRID =</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11029</td>
			<td>dilution = 1:200; RRID = AB_138404</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-Cy3</td>
			<td>Southern Biotech</td>
			<td>4050-02</td>
			<td>dilution = 1:200; RRID = AB_2795952</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-FITC</td>
			<td>Jackson Immunoresearch Laboratories</td>
			<td>711-165-152</td>
			<td>dilution = 1:200; RRID = AB_2307443</td>
		</tr>
		<tr>
			<td>Goat anti-rat Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11006</td>
			<td>dilution = 1:200; RRID = AB_2534074</td>
		</tr>
		<tr>
			<td>Goat anti-rat Alexa Fluor 647</td>
			<td>Thermo-Fisher</td>
			<td>A21247</td>
			<td>dilution = 1:200; RRID = AB_141778</td>
		</tr>
		<tr>
			<td>Periostin</td>
			<td>Santa Cruz</td>
			<td>SC67233</td>
			<td>dilution = 1:100; RRID = AB_2166650</td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>Sigma Aldrich</td>
			<td>V2258</td>
			<td>dilution = 1:200; RRID = AB_261856</td>
		</tr>
		<tr>
			<td>&#945;-smooth muscle actin (&#945;SMA)</td>
			<td>Sigma Aldrich</td>
			<td>A2547</td>
			<td>dilution = 1:1000; RRID = AB_476701</td>
		</tr>
		<tr>
			<td>Fibroblast specific protein 1 (FSP1)</td>
			<td>Millipore 07-2274</td>
			<td>07-2274</td>
			<td>dilution = 1:100; RRID = AB_10807552</td>
		</tr>
		<tr>
			<td>CD45 Magnetic Beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 Magnetic Beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-087-418</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-feeder cells-APC (MEFSK4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-102-900</td>
			<td>dilution = 1:100; RRID = AB_2660619</td>
		</tr>
		<tr>
			<td>anti-APC Beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-855</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG-APC</td>
			<td>Miltenyi Biotec</td>
			<td>130-103-034</td>
			<td>dilution = 1:100; RRID = AB_2661598</td>
		</tr>
		<tr>
			<td>Donkey anti-rat Alexa Fluor405</td>
			<td>Abcam</td>
			<td>ab175670</td>
			<td>dilution = 1:100</td>
		</tr>
		<tr>
			<td>anti-AN2/NG2</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-455</td>
			<td>dilution = 1:11; RRID = AB_2651235</td>
		</tr>
		<tr>
			<td>Other Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m Filter</td>
			<td>Thermo Scientific</td>
			<td>723-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm2 Cell Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pipet</td>
			<td>Fisherbrand</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Fisherbrand</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pipet</td>
			<td>Fisherbrand</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Tubes</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Edge Blade</td>
			<td>PAL</td>
			<td>62-0177</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-&#945;SMA Reporter Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separator Magnetic Field</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separation Column</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qaigen Rneasy Mini Kit</td>
			<td>Qaigen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion RNAqueous Micro Total Isolation Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad iScript cDNA Syntehsis Kit</td>
			<td>BioRad</td>
			<td>1708891</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30G Needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3 Laser Flow Cytometry Machine (BD LSRFortessa)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 Laser Flow Cytometry Machine (BD FACSAria III)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Data Acquiring Software (BD FACSDiva Software v8.0a)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Data Analysis Software (FlowJo Software)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Flow gating scheme demonstrating myofibroblast isolation using &#945;SMA-GFP reporter mice 
Uninjured hearts showed no detectable GFP+ cells in &#945;SMA-GFP reporter mouse model; hence, they were used to establish a gate for the background signal of the GFP channel post-compensation (Figure 2). &#945;SMA+ cells were sorted based on the presence of GFP expression from the injured left ventricle 10 days following MI. A small percentage of endothelia...

Watch this Scientific Journal Video about Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts at JoVE.com

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

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 ...

This protocol allows us to isolate both quiescent and injury-induced activated fibroblast populations with significant purity. The main advantage of this technique is its flexibility for improvisation. For flow, as well as bead-based isolation, one can incorporate antibodies against contaminating cells to exclude them from the isolated target cells.This technique is primarily used for cardiac fibroblast isolation. However, it can be utilized to isolate fibroblasts from other tissues. Demonstrating this procedure will be Meiling Melzer, an undergraduate intern, and David Beier, a research assistant from my laboratory.To prepare a six-well plate for storing six hearts during dissection, dispense two milliliters of cold KHB into each well, and place the plate on ice. Spray the body with 70%ethanol. Orient it so the ventral side is facing the experimenter.Pin the appendages to prevent interference. Without piercing the liver, cut open the abdominal skin and muscle. Cut vertically toward the sternum, carefully opening the thorax without piercing the heart.Next, continue to cut through the ribcage to expose the heart. Using forceps, gently lift the heart out of the chest, cutting away any lung or excess tissue attached to the outside of the heart. Separate the left ventricle.For each dissection, place the ventricle in a separate well of the six-well plate. First, use forceps to repeatedly squeeze and agitate the ventricle in KHB to remove excess blood. Transfer the ventricle to a clean, sterile, 10-centimeter-square plate.Next, using a single-edged blade, quickly mince the ventricle into small pieces. Add one milliliter of collagenase digestion cocktail, and continue mincing. When the pieces are small enough to transfer with a one-milliliter micropipette, transfer them to a 50-milliliter conical tube.Wash the plate twice with two milliliters of cocktail, and transfer the wash to the tube. Incubate the tube for 30 minutes, and resuspend the cells as described in the manuscript using a five-milliliter pipette. After the initial resuspension, incubate the cells for 15 minutes, and resuspend again using a 10-milliliter pipette.Then, place a 40-micrometer cell strainer on top of a new, 50-milliliter conical tube, and prime the strainer by wetting it with one to two milliliters of KHB. Add 25 milliliters of KHB to the digestion suspension, and resuspend. Filter the suspension through the strainer, replacing the strainer as needed.Centrifuge the filtrate at 400 times g and four degrees Celsius for 10 minutes. Remove the supernatant, and resuspend the pellet in 1x RBC lysis buffer using five milliliters of buffer per heart. After incubating the suspension for two minutes at room temperature, centrifuge the suspension at 400 times g for 10 minutes at room temperature.Remove the supernatant, and then resuspend the pellet in one milliliter of KHB. Then, add nine milliliters of KHB to the suspension. Filter the suspension through a primed, 40-micrometer cell strainer into a new, 50-milliliter conical tube.Centrifuge the conical tube at 400 times g for 10 minutes at room temperature. Finally, remove the supernatant, and resuspend the pellet in one milliliter of fibroblast media or PBS. After the cell number has been determined, fibroblasts can be isolated from the suspension by three different methods described in the manuscript.To begin isolation by differential plating, prepare a six-well plate by adding two milliliters of fibroblast media to each well. Swirl the plate to cover the well bottoms. If the mouse heart cells are suspended in PBS, centrifuge and resuspend them in one milliliter of fibroblast media per heart.Add one milliliter of cell suspension, the equivalent of one heart, to each well. Swirl the plate to evenly distribute cells. Add an additional one to two milliliters of fibroblast media per well for a total volume of up to four milliliters per well.Incubate at 37 degrees Celsius for four hours. Because the fibroblasts will selectively adhere to the wells, they can be isolated by removing and discarding the media. Wash the remaining attached cells with two milliliters of PBS, and then add two to four milliliters of sterile fibroblast media per well.Incubate at 37 degrees until confluent, changing the media every two to four days. To begin the magnetic labeling and separation of CD45-positive hematopoietic cells, centrifuge a suspension of mouse heart cells at 500 times g for five minutes. Remove the supernatant, and resuspend the cell pellet in one milliliter of equilibration buffer.Count the cells using a hemocytometer. After centrifuging the cells again, resuspend the cell pellet in equilibration buffer. Add CD45-positive magnetic beads, and mix well.Incubate for at least 15 minutes at four degrees Celsius. Wash the cells by adding equilibration buffer and centrifuging at 500 times g for 10 minutes at four degrees Celsius. Remove the supernatant, and count the cells using a hemocytometer.Resuspend the pellet in equilibration buffer. Pass the suspension through a 40-micrometer filter to prevent cell aggregations from clogging the separation column. Next, place a separation column in the magnetic field of a suitable separator, and equilibrate the column with at least three milliliters of PBS.Pour the cell suspension through the column, and collect the flow-through, which will contain the unlabeled cells, in a 15-milliliter conical tube. Wash the column three times with three milliliters of equilibration buffer, and combine the washes with the flow-through. Remove the column from the separator, and place the column on a 15-milliliter conical tube.To elute the labeled CD45-positive cells, pipette five milliliters of equilibration buffer into the column, and plunge the column firmly. Centrifuge the tube of eluent and the tube of flow-through at 500 times g. Count the cells using a hemocytometer.To complete the isolation, follow the protocols in the manuscript for the CD31-positive endothelial cells and the MEFSK4-positive fibroblasts, which are similar to the protocol for the CD45-positive cells. Fluorescence-activated cell sorting was performed on single cells isolated from alpha-SMA-GFP mouse hearts following myocardial infarction. A representative gating scheme demonstrates GFP-positive cells co-expressing CD31, CD45, or AN2.In the injured mouse hearts, a small percentage of endothelial cells and hematopoietic cells expressed GFP. However, GFP-positive/CD31-negative/CD45-negative cells did not express AN2, a pericyte marker. GFP-positive cells expressed alpha-SMA, collagen one alpha one, periostin, and vimentin.Uninjured fibroblasts isolated by selective adhesion expressed vimentin but did not demonstrate expression of alpha-SMA, periostin, or collagen one alpha one. Both uninjured and activated fibroblasts expressed the MEFSK4 antigen. Both uninjured fibroblasts, isolated by selective adhesion, and myofibroblasts, isolated and sorted from alpha-SMA-GFP mice, demonstrated an ability to contract collagen.It is important to mince the tissue thoroughly before incubation. Before bead-based separation, it is important to pass the suspension through a 40-micrometer filter to prevent cell aggregations. The cells purified by this method could be further purified using magnetic beads associated with pericyte-specific antibodies to ensure a high level of purity.With the availability of new bead-conjugated antibodies, as well as reporter transgenic mice, one can utilize these techniques to isolate and study distinct cellular populations, not just fibroblasts.

Research

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Biology

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

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

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

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

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

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

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...