We would like to thank the Flow cytometry Core Facilities at the University of Ottawa and the Keenan Research Centre for Biomedical Sciences (KRC), St Michaels Hospital Unity Health Toronto for their expertise and guidance in optimization of the flow cytometry/FACS protocol presented in this manuscript. This work was funded by Medicine by Design New Ideas 2018 Fund (MbDNI-2018-01) to JB.

Investigators conducting this protocol must receive permission from their local animal ethics board/care committee. All animal work was approved by the St. Michael&#39;s Hospital Unity Health Toronto Animal Care Committee (ACC #918) and was conducted in accordance with the guidelines set forth by the Canadian Council on Animal Care (CCAC). A schematic of the flow cytometry protocol is shown in Figure 1. If the downstream application is FACS and subsequent cell culture, all steps should be co...

An optimized, validated FAPs isolation protocol for rat muscle is essential for researchers who wish to study injury models that are not feasible in the mouse for biologic or technical reasons. For example, mice are not an optimal animal model in which to study chronic local, or neurodegenerative injuries such as long-term denervation. Biologically, the short lifespan and rapid aging of mice make it difficult to accurately delineate the muscle sequalae due to denervation from the confounding factor of aging. From a techn...

Fibro-adipogenic progenitor cells (FAPs) are a population of resident multipotent progenitor cells in skeletal muscle that play a critical role in muscle homeostasis, repair, and regeneration, and conversely, also mediate pathologic responses to muscle injury. As the name suggests, FAPs were originally identified as a progenitor population with the potential to differentiate into fibroblasts and adipocytes1&#160;and were purported to be the key mediators of fibro-fatty infiltration of skeletal muscle in chronic injury and disease. Further study revealed that FAPs are additionally capable of osteogenesis and chondrogenesis2,3,4. Thus, they are more broadly notated in the literature as mesenchymal or stromal progenitors3,5,6,7,8. In acute skeletal muscle injury, FAPs indirectly aid in regenerative myogenesis by transiently proliferating to provide a favorable environment for activated muscle satellite cells and their downstream myogenic progenitor (MPs) counterparts1,9,10. In parallel with successful regeneration, FAPs undergo apoptosis, returning their numbers to baseline levels1,9,10,11. In contrast, in chronic muscle injury, FAPs override pro-apoptotic signals, which results in their persistence9,10,11&#160;and abnormal muscle repair.
In vivo studies evaluating the cellular and molecular mechanisms by which FAPs mediate muscle responses have utilized murine animal models to date1,7,9,10,11,12,13,14. While genetically engineered mice are powerful tools for&#160;use in these analyses, the small size of the animal limits tissue availability for study in long-term localized injury models where muscle atrophy can be profound, such as traumatic denervation. Furthermore, measurement of muscle strength and physical function requires ex vivo or in situ measurements that necessitate termination of the mouse, or in vivo methods that require surgery and/or a general anaesthetic to permit evaluation of muscle contractile performance15,16,17,18,19,20. In rats, well validated and globally utilized muscle functional analyses, in addition to analyses for more complex motor behaviors such as gait analysis (e.g., Sciatic Function Index, CatWalk analysis) exist and are performed in awake and spontaneously moving animals21,22,23,24. This additionally optimizes the principles of minimal morbidity in animal experimentation, and numbers of research animals used. The rat thereby provides the FAPs investigator the added flexibility of greater injured muscle volume for protein and cellular analyses and the ability to undertake serial assessments of muscle complex static and dynamic functional activity and behaviors, in the alert animal.
FAPs have primarily been identified and isolated from whole muscle samples using flow cytometry and Fluorescence-activated cell sorting (FACS) respectively. These are laser-based assays that are able to identify multiple specific cell populations based on characteristic features such as size, granularity, and a specific combination of cell surface or intracellular markers25. This is highly advantageous in the study of an organ system such as skeletal muscle, as homeostasis and regeneration are&#160;complex, multifactorial processes coordinated by a plethora of cell types. A seminal study identified FAPs, as well as MPs, using flow cytometric methods in mouse skeletal muscle1. They demonstrated that FAPs are mesenchymal in nature, as they lacked surface antigens specific to cells from endothelial (CD31), hematopoietic (CD45), or myogenic (Integrin-&#945;7 [ITGA7]) origins, but expressed the mesenchymal stem cell marker Sca-1 (Stem cell antigen 1)1&#160;and differentiated into fibrogenic and adipogenic cells in culture. Other studies demonstrated successful isolation of mesenchymal progenitors in muscle based on the expression of an alternative stem cell marker, platelet-derived growth factor receptor alpha (PDGFR&#945;)2,7,8&#160;and further analysis revealed these likely to be the same cell population as FAPs3. FAPs are now commonly identified in flow cytometry using either Sca-1 or PDGFR&#945; as a positive selection marker1,9,10,11,12,13,14,26,27,28,29,30,31. The use of PDGFR&#945; is preferential for human tissue&#160;however, as a direct human homologue of murine Sca-1 has yet to be identified32. In addition, other cell surface proteins have been reported as markers of MPs (e.g., VCAM-1), providing a potential alternative to ITGA7 as an indicator of cells of myogenic lineage during FAPs isolation33.
While flow cytometry/FACS is a powerful methodology for studying the role and pathogenic potential of FAPs in skeletal muscle1,9,10,11,13,29, it is limited technically by the specificity and optimization of its required reagents. Since flow cytometric identification and isolation of FAPs has been developed and conducted in mouse animal models1,9,10,11,29, this poses challenges for researchers who wish to study FAPs in other model organisms. Many factors - such as optimal tissue size to be processed, as well as reagent and/or antibody specificity and availability - differ depending on the species used.
In addition to the technical barriers to studying FAPs in a novel animal model, they have largely been studied in an acute, toxic setting - usually via intramuscular chemical injection or cardiotoxin. Evaluation of the long-term dynamics of FAPs is limited primarily to assessment in Duchenne&#39;s muscular dystrophy, using the mdx mouse model9,10,11, and models of combination muscle injury such as massive rotator cuff tear where concurrent tendon transection and denervation is performed on shoulder musculature26,27,28. The response of FAPs to the sole insult of chronic traumatic denervation, a common occurrence in work-place accidents in heavy industry, agriculture, and in birth traumas (brachial plexus injury)34,35,36,37&#160;with significant morbidity, has not been as well characterized, often limited to a short-term time frame11,38.
We describe a method for identifying and isolating FAPs and MPs from healthy as well as severely atrophic and fibrotic skeletal muscle in the rat. First, identification of CD31-/CD45-/Sca-1+/VCAM-1- FAPs and CD31-/CD45-/Sca-1-/VCAM-1+ MPs using a tissue digestion and flow cytometry staining protocol is demonstrated and subsequent validation of our findings is performed through culture and immunocytochemical staining of FACS-isolated cells. Using this method, we also report a novel FAPs time-course in a long-term isolated denervation injury model in the rat.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL Polypropylene Round-Bottom Tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dishes</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate</td>
			<td>ThermoFisher</td>
			<td>130185</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm glass coverslips, No.2</td>
			<td>VWR</td>
			<td>89015-724</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>Beckton Dickenson</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>FroggaBio</td>
			<td>91014</td>
			<td></td>
		</tr>
		<tr>
			<td>20 gauge needle</td>
			<td>Beckton Dickenson</td>
			<td>305176</td>
			<td></td>
		</tr>
		<tr>
			<td>25mL Serological pipette</td>
			<td>Sarstedt</td>
			<td>86.1685.001</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL centrifuge tubes</td>
			<td>FroggaBio</td>
			<td>TB50</td>
			<td></td>
		</tr>
		<tr>
			<td>AbC Total Antibody Compensation Beads&#160;</td>
			<td>ThermoFisher</td>
			<td>A10497</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride, Reagent Grade</td>
			<td>Bioshop</td>
			<td>AMC303.500</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Conjugation Kit, 50-100&#181;g</td>
			<td>Biotium</td>
			<td>92307</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquatex Aqueous Mounting Medium</td>
			<td>Merck</td>
			<td>108562</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolaminin 411 LN</td>
			<td>Biolamina</td>
			<td>LN411</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Bioshop</td>
			<td>ALB001</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Bioshop</td>
			<td>CCL444.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Gibco</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Plus Absolute Counting Beads</td>
			<td>ThermoFisher</td>
			<td>C36995</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Millipore Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM) (1X)</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>(+)4.5 g/L D-Glucose 
			(+)L-Glutamine 
			(+)110 mg/L Sodium Pyruvate
			</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>FisherScientific</td>
			<td>S311</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSClean Solution</td>
			<td>Beckton Dickenson</td>
			<td>340345</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSDiva Software</td>
			<td>Beckton Dickenson</td>
			<td>--</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSRinse Solution</td>
			<td>Beckton Dickenson</td>
			<td>340346</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Sheath Fluid</td>
			<td>Beckton Dickenson</td>
			<td>342003</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>Beckton Dickenson</td>
			<td>--</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Medium</td>
			<td>Dako</td>
			<td>S302380-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 555 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21424</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 488 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 555 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21429</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Gibco</td>
			<td>16210-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F10 Media</td>
			<td>ThermoFisher&#160;</td>
			<td>11550043</td>
			<td>(+) Phenol Red 
			(+) L-Glutamine 
			(-) HEPES</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS) (1X)</td>
			<td>Multicell</td>
			<td>311-513-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Horse Serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Reichert</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, minimum 99.5% titration</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>ThermoFisher</td>
			<td>16050130</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Transforming Growth Factor &#946;1 (hTGF-&#946;1)</td>
			<td>Cell Signaling</td>
			<td>8915LF</td>
			<td></td>
		</tr>
		<tr>
			<td>Humulin R</td>
			<td>Lilly</td>
			<td>HI0210</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Millipore Sigma</td>
			<td>I5879</td>
			<td>Also known as 3-Isobutyl-1-methylxanthine</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td>Also known as 2-propanol</td>
		</tr>
		<tr>
			<td>Lewis Rat, Female</td>
			<td>Charles River Kingston</td>
			<td>004 (Strain Code)</td>
			<td>200-250 grams used</td>
		</tr>
		<tr>
			<td>LSRFortessa X-20 Benchtop Cytometer</td>
			<td>Beckton Dickenson</td>
			<td>--</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>EP-5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>MoFlo XDP Cell Sorter</td>
			<td>Beckman Coulter</td>
			<td>--</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-CD31::FITC Antibody</td>
			<td>Abcam</td>
			<td>ab33858</td>
			<td>Clone TLD-3A12</td>
		</tr>
		<tr>
			<td>Mouse Anti-CD45::FITC Antibody</td>
			<td>Biolegend</td>
			<td>202205</td>
			<td>Clone OX-1</td>
		</tr>
		<tr>
			<td>Mouse Anti-CD106::PE Antibody</td>
			<td>Biolegend</td>
			<td>200403</td>
			<td>Also known as VCAM-1</td>
		</tr>
		<tr>
			<td>Mouse Anti-MHC Antibody</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>N/A</td>
			<td>Also known as MF20</td>
		</tr>
		<tr>
			<td>Mouse Anti-Pax7 Antibody</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Buffered Formalin, 10 %</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Millipore Sigma</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Cy7 Conjugation Kit</td>
			<td>Abcam</td>
			<td>ab102903</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma&#160;</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, pH 7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>(-)Calcium Chloride 
			(-)Magnesium Chloride
			</td>
		</tr>
		<tr>
			<td>Potassium Bicarbonate, Reagent Grade</td>
			<td>Bioshop</td>
			<td>PBC401.250</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Fibroblast Specific Protein 1 (FSP-1) Antibody</td>
			<td>Invitrogen</td>
			<td>MA5-32347</td>
			<td>FSP-1 also known as S100A4</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Integrin-a7 Antibody</td>
			<td>Abcam</td>
			<td>ab203254</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Laminin Antibody</td>
			<td>Sigma</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Perilipin-1 Antibody</td>
			<td>Abcam</td>
			<td>ab3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Sca-1 Antibody</td>
			<td>Millipore Sigma</td>
			<td>AB4336</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Recombinant Anti-Collagen Type I Antibody</td>
			<td>Abcam</td>
			<td>ab260043</td>
			<td>Also known as Col1a1</td>
		</tr>
		<tr>
			<td>Rabbit Recombinant Anti-PDGFR Alpha Antibody</td>
			<td>Abcam</td>
			<td>ab203491</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic</td>
			<td>Gibco</td>
			<td>PHG0266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td></td>
		</tr>
		<tr>
			<td>Troglitazone</td>
			<td>Millipore Sigma</td>
			<td>T2573</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bioshop</td>
			<td>TWN510</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification, isolation, and characterization of fibro-adipogenic progenitors (faps) and myogenic progenitors (mps) in skeletal muscle in the rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identifying FAPs and MPs via flow cytometry using a novel antibody panel including Sca-1 and VCAM-1 
The gating strategy for identifying FAPs in rat muscle is based upon flow cytometry protocols in the mouse29, which gate on CD31 (endothelial) and CD45 (hematopoietic) positive cells (termed the lineage [Lin]) and examines the fluorescent profile of FAPs marker Sca-1 and MPs marker ITGA7 from the linage-negative (Lin-) populat...

Watch this Scientific Journal Video about Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat at JoVE.com

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

As FAPs are mediators of muscle regeneration and pathological fibrosis, our protocol allows for characterization of FAP dynamics post muscle injury and isolation of FAPs for in vitro or ex vivo studies. To date, studies have been performed exclusively on FAPs isolated from mice. Our protocol enables effective isolation of FAPs from the larger rat, providing much greater tissue availability for downstream assays.Prolonged tissue processing time can negatively impact FAPs viability. If multiple samples are being processed simultaneously, it is recommended that two operators perform the protocol in tandem to maximize isolated cell viability. Begin by placing the harvested muscle tissue in a sterile 10-centimeter cell culture dish.Gently tear and mince the tissue with forceps and remove connective tissue to obtain approximately three to four-millimeter cubed pieces. Transfer the minster tissue to a sterile 50-milliliter conical tube containing six milliliters of DMEM and 1%penicillin-streptomycin. Next, activate 365 microliters of collagenase II solution by adding 10 microliters of 300-millimolar calcium chloride solution.Add the activated collagenase II solution to the tissue slurry for a final concentration of 250 units per milliliter. Incubate the tubes in a shaker for one hour and at 37 degrees Celsius at 240 x g with manual agitation every 15 minutes to dislodge tissue adhered to the side of the tube. After one hour of incubation, add 100 microliters of collagenase II and 50 microliters of dispase to the sample.Then, pipette samples 15 to 20 times with a serological pipette until the solution is homogenous. Incubate the sample again for 30 minutes at 37 degrees Celsius and 240 x g with manual agitation every 15 minutes. Slowly shear the muscle solution samples through a 20-milliliter syringe with a 20-gauge needle for 10 cycles.Then, place a 40-micron cell strainer on a sterile 50-milliliter conical tube and wet it by pipetting five milliliters of DMEM supplemented with 10%FBS and 1%penicillin-streptomycin. Pipette the sample one milliliter at a time through the strainer. Once the whole sample is filtered, wash the cell strainer with DMEM supplemented with 10%FBS and 1%penicillin-streptomycin to bring the total volume of sample to 25 milliliters.Split the sample volume equally into two 15-milliliter conical tubes and centrifuge at 15 degrees Celsius, 400 x g for 15 minutes. After centrifugation, aspirate the supernatant and resuspend the pellet in one milliliter of RBC lysis buffer at room temperature for seven minutes. Then, add nine milliliters of wash buffer to bring the volume to 10 milliliters and centrifuge at 15 degrees Celsius, 400 x g for 15 minutes.After centrifugation, aspirate the supernatant and resuspend the pellet in one milliliter of wash buffer. Transfer an appropriate volume of cells to a separate 1.5-milliliter microcentrifuge tube and mix it with trypan blue dye. Count live cells on a light microscope using a hemocytometer.For flow cytometry, transfer one to 2 million cells per experimental sample to a sterile 1.5-milliliter microcentrifuge tube. Bring the sample volume to one milliliter with wash buffer, and place the tube on ice. Set up all controls for the experiment.For cell controls, aliquot 500, 000 to 1 million cells in one milliliter of wash buffer in a 1.5-milliliter microcentrifuge tube and place it on ice. If the experiment is being performed for the first time, single-stained cell suspensions should also be included. To set up bead controls, add around 150, 000 positive compensation beads to each labeled 1.5-milliliter centrifuge tube.Prepare the viability control by transferring half of the cell volume from the viability tube to refresh 1.5-milliliter microcentrifuge tube labeled dead. Incubate the dead tube at 65 degrees Celsius for two to three minutes, then place it on ice. After the incubation, return the dead cells to the viability tube.Centrifuge the single-cell suspensions, including experimental and control samples at 500 x g and four degrees Celsius for five minutes, and resuspend the cell pellets in 100 microliters of wash buffer after aspirating the supernatant. Add antibodies according to control or experimental condition, and gently flick the sample to ensure complete mixing. Then, incubate them on ice in the dark for 15 minutes.For compensation beads, incubate the tube at room temperature in the dark for 15 minutes. Bring the volume of each sample to one milliliter by adding 900 microliters of wash buffer to the single-cell suspension, and 900 microliters of PBS for compensation bead controls. Centrifuge single-cell suspensions at 500 x g and four degrees Celsius for five minutes, and compensation bead controls at 300 x g and four degrees Celsius for five minutes.After centrifugation of the samples, aspirate the supernatant and resuspend the cell pellet in 300 microliters of wash buffer, and bead controls in 300 microliters of PBS and 150, 000 negative compensation beads. Keep the cell samples on ice and bead samples at room temperature under aluminum foil. Proceed with flow cytometry acquisition by setting up the gating strategy to identify fibro-adipogenic progenitors and myogenic progenitors.In the representative analysis, successful conjugation of the Sca-1 APC Antibody with single staining of compensation beads and cell suspensions generated from healthy rat gastrocnemius muscle was confirmed. Five different concentrations of Sca-1 APC were titrated on single-cell suspensions, and the optimal concentration of antibody was identified based on greatest fluorescence intensity with minimal background staining. Flow cytometric identifications of FAPs and MPs in rat gastrocnemius was performed using the gating strategy shown here.Samples were first gated to exclude debris in counting beads. Cells were then gated to exclude doublets by both front scatter and side scatter characteristics. The viability of resulting cells was assessed by staining with SYTOX Blue.SYTOX Blue negative singlets were assessed for CD31 and CD45 to exclude Lin+fractions. The Lin-population was assessed. Cells that were single positive for Sca-1 were designated FAPs, and cells that were single positive for VCAM-1 were designated MPs.With co-immunostaining immediately after sorting, the freshly isolated population of FAPs displayed positive staining for PDGFRalpha with no contamination by Pax7 positive cells. Conversely, the sorted population of MPs stained positive for Pax7 with an absence of PDGFRalpha-positive cells. FAPs demonstrated differentiated fibroblasts and adipocytes at day 12 in adipogenic differentiation media with expression of fibroblast-specific protein 1 and perilipin 1, respectively.The presence of neutral triglycerides and lipids from mature adipocytes was evident with Oil Red O staining. Additionally, the FAPs demonstrated a presence of collagen type I and an absence of contaminating myocytes. MPs grown in myogenic differentiation media on day 12 displayed the presence of mature myocytes and fused multinucleated myotubes, and were clear of fibroblasts and adipocyte contamination.When using this technique to isolate live cells for long-term culture, researchers must ensure to use aseptic technique while simultaneously working efficiently to ensure good cell viability.

identification-isolation-characterization-fibro-adipogenic

Research

JoVE Journal

Biology

5.4K vistas.  St Michaels Hospital, Unity Health Toronto. Como los FAPs son mediadores de la regeneración muscular y la fibrosis patológica, nuestro protocolo permite la caracterización de la dinámica de faP post lesión muscular y el aislamiento de FAPs para estudios in vitro o ex vivo. Hasta la fecha, los estudios se han realizado exclusivamente en FAPs aislados de ratones. Nuestro protocolo permite el aislamiento efectivo de los FAPs de la rata más grande, proporcionando una disponibilidad de tejido mucho mayor para los ensayos posteriores.