In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.
In cardiac myocytes a complex network of membrane tubules – the transverse-axial tubule system (TATS) – controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
In gezonde dwarsgestreepte spiercellen, buisvormige membraan structuur met "dwars" oriëntaties (T-tubuli) loodrecht op de hoofdleiding cel as zijn overvloedig. Dientengevolge hebben T-tubuli gekenmerkt als continue uitbreidingen van de spiercel main "lateraal" oppervlak membraan (sarcolemma), die diep doordringen in de cytosol naar het celcentrum. De fysiologische rol van T-tubuli continu met het buitenoppervlak membraan snelle elektrische koppeling van externe intracellulaire compartimenten gevormd door SER organel contact domeinen gehele relatief grote hartspiercel volume nanometrische nabijheid koppeling van voltage-geactiveerde L-Ca2 + kanalen (Cav1.2) naar binnen (I Ca) activeren van aangrenzende ryanodinereceptor (RyR2) SER Ca 2 + releases. In hartspiercellen (VM's), de niet-continue membraan contacten ("knooppunten") tussen de junctionele SER domeinen en T-tubules wordt gedacht dat duizenden individuele intracellulaire Ca2 + afgifte nanodomains controle in elke cel 1.
Voor elk contact domein, de naast elkaar geplaatste gedeelten membraan elk van de T-tubuli en perifere (junction) SER ongeveer 15 nm dicht bij elkaar, dus gedefinieerd als nanodomain. Daarbij worden zeer kleine individuele cytoplasma deelruimten gescheiden die quasi-cel-autonome compartiment gedrag mogelijk te maken. Bij een inkomende actiepotentiaal activeert Cav1.2 kanalen in de T-tubuli van VM's, een relatief kleine Ca 2 + naar binnen lopende zal snel toenemen de deelruimte Ca 2 +-concentratie [Ca 2 +] S in de attoliter formaat nanodomain 1. Vervolgens wordt de [Ca 2 +] S toename activeert Ca 2 + -Gated ryanodine receptoren (RyR2) binnen nanometer nabijheid in het naast elkaar SER membraan knooppunt, en deze koppeling proces gebeurt in alle elektrisch paard myocyte nanodomains. RyR2s voorkomen als dichte multikanaals clusters voor een stoïchiometrie van 1 Cav1.2 kanaal 5-10 RyR2 kanalen 2. Aangezien de SER naar cytosol [Ca2 +] gradiënt zeer steil (10 verhouding 4: 1) en RyR2s gefunctioneerd als high-geleiding Ca2 + afgifte kanalen functioneel gekoppelde clusters, RyR2 activatie resulteert in een kwantitatief grote Ca2 + los stroom van T-tubulus gekoppeld junctional SER domeinen toenemende lokale deelruimte [Ca 2 +] S tot 100 uM of hoger binnen 1-2 msec 3,4. Dit cardiale signaal amplificatie gedrag wordt ook wel Ca2 + geïnduceerde Ca2 + afgifte (CICR). Samen genomen, de T-tubuli zijn essentieel membraan structuur die snel activeren Ca 2 + vrijlating signalen via junctional nanodomain SER contacten en cel-breed CKP tijdens excitatie-contractie (EC) koppeling.
Naast T-tubuli axiaal buisjes (A-tubules) met een significant verschillende oriëntatie parallel aan de hoofd (langs) as cellen werden gedocumenteerd door elektronenmicroscopie (EM), confocale en 2-foton microscopie studies. Bijvoorbeeld, een cel gehele continue rooster van A-tubules tussen myofibrils verbonden met T-tubuli dichtbij sarcomeer Z-lijnen werd getoond door extracellulaire tracers en EM beeldvorming van vaste cavia VM 5. Met behulp van extracellulaire-dextran gekoppeld fluoresceïneverkleuring en live 2-foton beeldvorming van rat VM's, werd een complex reticular 3D tubulus netwerk gevisualiseerd bestaande uit ~ 60% T-tubuli en ~ 40% A-buisjes 6. Deze studie niet alleen geleid tot 3D visualisatie van overvloedige A-tubules, maar ook tot het besef dat snijden voor EM visualisatie is inherent beperkt de analyse van complexe en dynamische membraan netwerken zoals de dwarse coaxiale tubulus systeem (TATS). Bijgevolg confocale live cell imaging van TATS membranen direct gekleurd met di-8-ANNEPS werd ontwikkeld. Als levende cellenTATS netwerken worden geanalyseerd door Fourier transformatie, wordt de regelmatige verschijning van T-tubulus componenten in de ruimte in de buurt van sarcomeer Z-lijnen weerspiegeld door het ensemble vermogensspectrum van een regio van dwarsgestreepte signalen 7. Deze indirecte analyse strategie werd gebruikt om cel-grote regionale veranderingen in TATS component regelmatigheid detecteren ziektemodellen 7. Zo shRNA gemedieerde junctophilin-2 afbraakprijzen veroorzaakt hartfalen en de isovorm specifiek eiwit tekort resulteerde in T-tubulus reorganisatie met nanodomain Ca 2 + vrijlating dysfunctie 8. We hebben onlangs uitgebreid de analyse van TATS membraan netwerken door middel van directe kwantitatieve benaderingen en verder door levende cellen superresolutiemicroscopie van individuele TATS componenten in de muis VM's met behulp van gestimuleerde emissie uitputting (STED) nanoscopie 9. Nanometrische beeldresolutie toegestaan voor een directe analyse van kleinere individuele TATS componenten, die een 50:50 verdeling van transversale benaderingversus axiale tubulus oriëntaties, kwantitatief bevestigen twee overvloedige nog verschillend georiënteerde individuele TATS componenten in gezonde muizen harten 9. Deze strategieën zullen verder worden beschreven in de rubriek onderstaande protocol.
Terwijl de fysiologische rol van de overvloedige A-tubulus componenten in het volwassen hart raadselachtig is gebleven, hebben EM studies SER membraan structuren geassocieerd met A-buisjes suggereert endogene Ca 2 + vrijlating nanodomains in cavia en rat VMs 5,10 gedocumenteerd. Confocale analyse van Cav1.2 en RyR2 vond een hoge colocalization op A-tubulus kruispunten 10. Aangezien ~ 20% spontane Ca2 + vonken in rat VM ontstaan relatief ver van Z-lijn strepen waarbij T-tubuli typisch voorkomen, is een argument is dat A-tubulus geassocieerde nanodomains inderdaad bestaan en functioneren als Ca 2 + versieplaatsen 11,12. Interessant is dat T-tubulus vorming en rijping occurs pas na de geboorte en loopt parallel met de groei van cardiale cellen, bijvoorbeeld door het kiemen van voorloper sarcolemmale invaginations op P5 en onvolwassen vertakt TATS netwerk samenstellingen bij een P10 bij muizen 13. Het blijkt dat Junctophilin-2 is bijzonder belangrijk voor postnatale maturatie TATS netwerk aangezien shRNA neerhalen verhinderde de verankering van T-tubuli membranen SER knooppunten leidt tot vertraagde Ca2 + afgifte en pathologische TATS organisatie overeenstemming met onrijpe A-tubulus gedomineerd architecturen VM 13. Deze waarnemingen kunnen uiteindelijk leiden tot proof-of-concept dat T-buisjes te vormen door middel van membraan invaginatie processen, terwijl A-buisjes kan morph door aanvullende of zelfs alternatieve intracellulaire mechanismen 14.
Karakterisering van TATS membraan veranderingen in hart-en vaatziekten is uitgegroeid tot een belangrijk onderzoeksgebied voor pathofysiologische vragen. De eerste berichten in een hond model van pacing-geïnduceerde hart failure toonde een verlies van T-tubuli en Cav1.2 (I Ca) 15. Een varken van ischemische cardiomyopathie vertoonden verminderde T-tubulus dichtheden en een verminderd synchronie van intracellulaire Ca 2 + los 16. Met behulp van een spontaan hypertensieve rat (SHR) van hartfalen, een verlies van T-tubuli werd geassocieerd met een verminderde nanodomain koppeling van Cav1.2 en RyR2 door het voorgestelde mechanisme van "RyR2 orphaning" 7. Een verlies van T-tubuli is ook aangetoond in humane VM ischemische, verwijde, en hypertrofische cardiomyopathie monsters 17. Bovendien, een toename A-buisjes werd gerapporteerd in weefselcoupes van menselijke gedilateerde cardiomyopathie 18. Na een myocardinfarct, toonden we een differentiële mechanisme van TATS reorganisatie in muis VM's met een significante afname van de T-tubuli in tegenstelling tot een toename van de A-tubulus onderdelen 9. Belangrijker nog, verbeterde lokale membraan contrast bereikt door levende cellen superreoplossing STED microscopie ingeschakeld gedetailleerde kwantitatieve elementanalyse door directe metingen, die aanzienlijke proliferatie van de A-tubuli met de algemene toename van de TATS netlengte en vertakking complexiteit 9 is. Bovendien werd aangetoond dat de training mag de T-tubulus remodeling in ratten te keren na een hartinfarct 19 en dat cardiale resynchronisatietherapie kan leiden tot een herinrichting van de T-tubuli te keren bij honden met atriale tachypacing geïnduceerd hartfalen 20. Samen genomen, zullen studies zowel in de zieke mens en dier VMs alsook mogelijke therapeutische interventies misschien wel profiteren van hoge kwaliteit celisolatie procedures en gedetailleerde kwantitatieve analyse strategieën zoals beschreven in het protocol en de resultaten hieronder secties.
Zoals onlangs aangetoond door mantelvlak versus TATS membraantransport van KATP kanaal isovormen 21, is het belangrijk om atriale m overwegenyocytes (AMS) als biologisch te onderscheiden, alsmede vergelijkende cardiale cel model versus VM's. T-buisjes werden onlangs gedocumenteerd bij schapen en menselijke AMs 22. Huidige gegevens wijzen erop dat enkele T-tubuli bestaan in AM cellen en meestal in grotere zoogdieren als schapen en mensen, maar niet in de kleine knaagdieren 23. In tegenstelling tot VM, in AMs intracellulaire Ca2 + afgifte blijkt plaats te vinden van het celoppervlak teeltmateriaal door diffusie naar het celcentrum waardoor aangegeven ruimtelijke en temporele Ca2 + gradiënten 23. In dit kader lijkt het van belang om de mechanismen van intracellulaire Ca 2 + signalering instabiliteit voor veel voorkomende ziekte vormen verhelderen zoals atriumfibrilleren 24. Samengevat, zijn zowel AM VM celisolatie en elk voor gezonde en zieke harten algemeen gebruikt protocollen. Alleen als celisolatie goed uitgevoerd zoals beoordeeld door microscopische documentatie voldoende cel kwaliteit, moet AM en VM monsters ca zijnrried voorwaarts voor kwantitatieve TATS analyse. Bijgevolg is de volgende protocol secties sterk afhankelijk zijn van hoge kwaliteit mobiele isolaten van muis of andere soorten, gevolgd door live-cell microscopie intact TATS membranen te analyseren. Zoals reeds eerder op gewezen, karakterisering van TATS membranen is een uitdagend onderzoeksgebied met een neiging tot fixatie en voorbereiding artefacten 6, membraan veranderingen als gevolg van osmotische veranderingen, en resolutie beperkingen van conventionele lichtmicroscopie 9. We merken op dat de recente state-of-the-art-protocollen voor de isolatie van de mens AMs voor Ca 2 + imaging en patch-clamp en rat VMs voor celkweek zijn eerder gepubliceerd in dit tijdschrift 25,26.
Although cardiac myocytes have been isolated and studied for decades32, a recent review concluded that consistent high-quality myocyte cell isolations remain challenging27. This reflects relatively complex protocols for isolation of primary cardiac myocytes vis-à-vis a lack of common standard approaches, shared metadata, and transparent cell quality documentation. Cell isolation protocols are usually customized by individual groups, produce variable outcomes of cell isolates, depend on individual model settings (e.g., species, age, coexisting heart conditions), and are usually adjusted for particular experimental conditions. Within the context of the quantitative TATS membrane studies and protocols presented here, an essential level of quality assessment and documentation concerns the confocal or superresolution microscopy of individual cell membrane structures prone to metabolic and isolation protocol dependent changes, both in AM or VM. Importantly, even if high yields of cell isolates suggest healthy intact myocytes, investigators need to document and critically judge each individual cell carefully against morphological criteria of surface and TATS membrane integrity versus non-specific damage due to isolation procedures versus specific changes due to different types of interventions as compared to control conditions. An important variable during cardiac cell isolation is the specific activity of a given collagenase lot. To select a new lot of collagenase, the enzymatic activity of several collagenase samples should be tested against each other by evaluating cardiac myocyte yield and quality, and according to manufacturer instructions. Ideally, a new lot of collagenase is identified with collagenase activity similar to previous successfully used lots (for extended evaluation of possible enzyme activities refer to the “collagenase lot selection tool” in the materials and methods table). Taken together, quantitative approaches of TATS membrane visualization depend critically on cell isolation quality and, vice versa, cardiac cell isolations leading to unspecific membrane damage as documented by TATS microscopy should trigger critical review and correction of the isolation procedures. Since cell isolation quality and TATS membrane visualization and quantification are intrinsically linked, the protocols discussed in this article cover all major aspects as a continuous strategy.
An additional challenge and common issue of cardiac studies, cell damage and/or cell loss occur due to metabolically compromising interventions e.g., following myocardial infarction9, yet need to be judged against potential inadvertent damage e.g., following unnoticed air embolism during cell isolation. Isolation of cardiac myocytes from diseased hearts may lead to additional, significant cell loss and decreased cell yields. Therefore, comparison of the total number of isolated intact cells between control and diseased hearts can be meaningful if cell isolation and counting are consistently applied through standardized protocols. Consequently, it is critical to judge cell integrity through an appropriate control group, which reflects the best possible myocyte cell isolation quality. Importantly, individual cell quality and live cell microscopy of healthy versus diseased versus myocytes inadvertently damaged by the isolation procedure may significantly influence the analysis of TATS membrane networks. The protocols presented here therefore emphasize the integrity and stability of physiological membrane components during cell isolation and live cell microscopy of intact membranes. The entire workflow is designed as a continuous strategy to achieve and preserve intact TATS membrane components while excluding damaged cells, since these will exhibit isolation dependent membrane artifacts like disrupted membrane tubules, membrane blebs, and altered TATS networks erroneously under control conditions and compromise further quantitative analysis. Vice versa, the same strategies are crucial for intervention studies with the potential to disrupt TATS membranes, which depend critically on meaningful comparison controls between true healthy versus true diseased cell with TATS membrane changes.
In addition, we address procedures to achieve the technically much more challenging isolation of AM cells. Despite progress and improved protocols, it is important to emphasize that it is not trivial to reproduce high quality cell isolations of VMs and even less reliable for AMs. This is due to the overall lower yield of AM cells where even small errors or variations during cell isolation may lead to complete failure of AM cell isolation, whereas a mild degree of VM cell damage might be less apparent in cell suspension due to relatively high cell numbers compared to AM. Since AM cells might become curved after isolation, analysis through several ROIs can be advantageous as outlined under step 4.3. Following a detailed procedure of cell isolation steps, we provide a protocol for direct integral membrane staining and confocal or STED superresolution imaging of TATS networks both for VMs and AMs. These protocols enable both quantitative analysis and differentiation of select components of TATS membranes through previously established parameters. As compared to VMs, the 3D organization and functional behaviors of the atrial TATS network in AMs are currently less understood.
The procedures to image TATS membranes in living cells (steps 3.1 to 3.7) were developed with commercial confocal (Table of Materials/Equipment) and custom-made STED fluorescence microscopes9. To optimize the microscope settings for fluorescent image generation and quantitative TATS analysis, the following points are of general importance:
In contrast to the direct analysis strategies presented here, previous publications describing TATS membranes and disease related changes, have used regional aggregate readouts of T-tubule density as quantitative strategy16,17, or indirect regional strategies based on Fourier transformation analysis of striated membrane signals in order to assess T-tubule component regularity7. In contrast, the quantitative approaches described here are directly related to individual TATS components and provide a number of additional parameters including membrane network properties and specific components like the percentage of A-tubules. Furthermore, the TATS network density can be quantified as the normalized length of the entire extracted skeleton per ROI area. The number of triple junctions of three individual, continuously connected tubule components can be used as a measure of the branching complexity of the TATS membrane network. We note that any analysis of smallest TATS components depends on the staining procedures. In our experience, 800 µl of a 50 µM di-8-ANEPPS solution are sufficient to stain complete TATS networks in a cell pellet containing 50,000 VM cells9. However, if the cell pellet contains a lower number of cardiac myocytes, if powerful fluorescence detectors are available, and if confocal imaging of the overall TATS network distribution rather than smallest membrane details and quantitative changes are of interest, lower dye concentrations may be used based on empirical testing. Finally, a software macro written for the described analysis can be used to automate the image processing steps to facilitate analysis of larger datasets, which is particularly useful for comparison between different treatment groups (e.g., drugs), cell types (e.g., AM versus VM), and pathophysiological interventions (e.g., sham versus myocardial infarction).
For image analysis of TATS networks, the following sequence of principle steps is applied: 1) rolling ball background–subtraction (4.5.1) to remove spatial variations in background intensity; 2) local contrast enhancement (4.5.2.); 3) image smoothing (4.5.3); 4) statistical region merging (4.5.4); 5) defining the threshold of image binarization (4.5.6); and 6) calculation of the skeleton data (4.5.8). A critical step during the skeletonization of fluorescent TATS images is the image binarization shown in Figure 6. The associated thresholding steps ultimately define which true membrane structures are detected to represent the underlying TATS components versus potentially false structures identified by error from background noise. Identification of the correct threshold for binary image analysis should correspond with the true TATS membrane structures, which depends on a sufficiently high signal-to-noise (SNR) ratio each for confocal and superresolution microscopy approaches. Therefore, a sufficient image quality should be established first and subsequently combined with critical judgment of individual cell quality including documentation by bright field images as outlined. Alternative options to adapt the image segmentation protocol for a given microscope data output and/or physiological questions include image deconvolution and other thresholding procedures like “Otsu” or “Iso-data” available as ImageJ plugins. Regardless of the final segmentation procedure, we consider the comparison between extracted and raw data by image overlay a mandatory quality control step. In summary, morphological and membrane integrity of individual isolated myocytes, sufficient staining of intracellular TATS membranes, parameter optimization for fluorescence imaging, and overlay control of extracted skeleton data will all contribute to the quality of fluorescent TATS images and quantitative results.
If larger species than mouse are used for cell isolation, the protocols can be readily adapted as appropriate. For the next larger species, rat hearts can be cannulated with a blunt 14 G cannula (outer diameter 2.1 mm) and perfused at 8 ml/min. Significantly older or diseased hearts may require even larger cannula sizes. In general, cardiac perfusion may be conducted either by constant pressure e.g., using a 1 m high water column between reservoir and aorta or by constant flow using a peristaltic pump. For cell isolation from small rodent hearts like mice and rats constant flow may be advantageous since collagenase digestion will eventually disrupt coronary resistance vessels leading to excessive perfusion rates from leaking vessel beds which will be controlled to some degree by constant flow protocols. In contrast, constant pressure perfusion is advantageous if monitoring of the flow rate and correct cannulation are a priority, which is advantageous for intervention models with altered blood vessel resistance behaviors as well as for training of the cell isolation procedures.
As outlined above, sufficient cell quality is very important for quantitative studies of endogenous membrane systems. However, during heart perfusion and collagenase digestion numerous factors can critically affect the quality of the cell isolation, which should never be underestimated during protocol optimization or trouble shooting27. In particular, the activity of a given collagenase lot should be determined for the specific tissue of interest e.g., atria or ventricles prior to execution of the experimental bona fide studies to establish isolation conditions to be maintained throughout the remainder of the study. Furthermore, the water quality, pH, temperature, optimization and cleaning of the perfusion setup will minimize the risk of inadvertent damage from contaminants and emboli, and potentially additional factors have to be monitored to establish optimal homeostatic conditions during cell isolation. BDM (2,3-butanedione-monoxime) a reversible inhibitor of myosin ATPase cross-bridges is commonly used during tissue dissection and digestion to sustain cardiac muscle relaxation, which increases the yield of cell isolations. Nevertheless, investigators need to be aware that BDM may exert non-specific phosphatase activities leading to off-target effects e.g., inhibition of Na+/Ca2+ exchange currents under certain conditions33. For some experiments it might be advantageous to replace BDM by blebbistatin as cardioplegic solution, an inhibitor with a high affinity for myosin at micromolar concentrations which is, however, toxic and relatively expensive and may have other off-target effects. Resting healthy cardiomyocytes should not show any contractions in the absence of electrical stimulation and such cells should be excluded from further analysis. On the other hand, cardiac myocyte contraction and relaxation in response to electrical stimulation at physiological extracellular Ca2+ concentrations can be used to establish normal contractile behavior as an additional measure to assess functional cell quality and/or abnormal behavior in heart disease versus healthy control cells.
In summary, the protocols for single cell isolation and quantitative image analysis described here have been successfully applied for confocal and superresolution microscopy of the TATS membrane network in VM9 and AM cells21 as well as for quantitative analysis of microtubule networks in fixed cardiac myocytes (data not shown). These and future applications of the protocols may open avenues for a variety of experimental questions such as the characterization of TATS membranes at different developmental stages or the analysis of membrane associated protein or organelle structures that contact the TATS network to exert highly localized, domain specific signaling functions in AM and VM cells.
The authors have nothing to disclose.
This work received support through Deutsche Forschungsgemeinschaft SFB 1002 (subprojects A05 and B05 to S.E.L.) and KFO 155 (subproject 4 to S.E.L.), a Halbach Foundation award to S.E.L. supporting E.W.; a grant from the German Cardiac Society to S.B.; and a DAAD exchange program supporting T.K. as visitor at the University of Maryland. The research leading to these results has received funding from the European Community’s Seventh Framework Program FP7/2007-2013 under grant agreement No. HEALTH-F2-2009-241526, EUTrigTreat (to S.E.L.). S.E.L. is a principal investigator of the German Center of Cardiovascular Research (DZHK).
Chemicals and enzymes | |||
2,3-Butanedione monoxime | Sigma-Aldrich, Munich, Germany | B0753 | |
Bovine calf serum | Thermo Scientific, Schwerte, Germany | SH30073 | Triple 0.1 µm sterile filtered. |
CaCl2 | Sigma-Aldrich, Munich, Germany | 21115 | Diluted 1:10 in MQ water to obtain 100 mM CaCl2 stock concentration. |
Collagenase type II | Worthington via Cell Systems, Troisdorf, Germany | on request | Enzymatic activity depends on individual collagenase batches. Collagenase II and other enzyme activities (Caseinase, Clostripain, Tryptic) can be assessed in the "collagenase lot selection tool". Determine cell yield and quality individually for each new lot of collagenase. |
Glucose | Carl Roth, Karlsruhe, Germany | HN06.1 | |
Heparin | Rotexmedica, Trittau, Germany | PZN-03862340 | Diluted in 0,9 % NaCl and injected subcutaneuosly in abdominal skin. |
HEPES | Carl Roth, Karlsruhe, Germany | 9105.4 | |
Forene 100% (V/V) | Abbott, Libertyville, IL, USA | B506 | Active agent: isoflurane, 250 ml. Use approximately 2 Vol% in air/oxygen dispenser instrument. |
KCl | Carl Roth, Karlsruhe, Germany | 6781.3 | |
KH2PO4 | Carl Roth, Karlsruhe, Germany | 3904.2 | |
Laminin (2 mg/ml) | BD Biosciences, Heidelberg, Germany | 354232 | Lamination is described under step 2.1. |
MgCl2 · 6 H2O | Carl Roth, Karlsruhe, Germany | 2189.2 | |
MgSO4 · 7 H2O | Carl Roth, Karlsruhe, Germany | 8283.2 | |
Na2HPO4 · 2 H2O | Carl Roth, Karlsruhe, Germany | 4984.2 | |
NaHCO3 | Carl Roth, Karlsruhe, Germany | HN01.1 | |
Taurin | Carl Roth, Karlsruhe, Germany | 4721.2 | |
Dyes | |||
Di-8-ANEPPS | Molecular Probes, Life Technologies, Darmstadt, Germany | D-3167 | Stock solution 2 mM in DMSO |
Trypan blue | Sigma-Aldrich, Munich, Germany | T8154 | Trypan blue is gently mixed 1:1 via tip-cut 1 ml plastic pipette with cell suspension prior to cell counting in Neubauer cytometer. |
Langendorff perfusion setup | |||
Circulation thermostat | Lauda, Lauda-Königshofen, Germany | Please refer to Louch et al. (JMCC 2011). Heat up thermostat und buffers in perfusion tubing to 37 °C 15 min prior to use. | |
Flexible silicone tubing Tygon for peristaltic pump | VWR, Darmstadt, Germany | 224-2252 | Tubing needs to be changed regularly. |
Flexible silicone tubing Tygon for thermostat | VWR, Darmstadt, Germany | 228-4340 | |
Heating coil surroundung perfusion tubing | Rettberg, Göttingen, Germany | custom-made | Heating coil and tubing needs to be cleaned thoroughly via MQ water after using. Do not use detergents. Glass components should be bathed regularly in 10 mM NaOH overnight. |
Peristaltic pump | Ismatec, Wertheim, Germany | ISM830 | |
Three way stop cock Discofix C Luer Lock 10 cm | Braun, Melsungen, Switzerland | 16500C | |
Three way stop cock Discofix 3SC | Braun, Melsungen, Switzerland | 4095146 | |
Instruments | |||
42 mm glass coverslips | Menzel Gläser via Thermo Scientific, Schwerte, Germany | on request | 0.13 – 0.16 mm thickness |
Cannula 21G | Becton, Dickinson and Company, Franklin Lake, NY, USA | 304432 | Cut to a length of ~ 5 mm, roughened with sandpaper. |
Coverslips for Neubauer cytometer 24 x 24 mm | Menzel Gläser via Thermo Scientific, Schwerte, Germany | on request | 0.38 – 0.42 mm thickness |
Graefe forceps, 0.5 mm tips, slight curve | Fine Science Tools, Heidelberg, Germany | 11151-10 | |
LSM 710 NLO | Carl Zeiss, Jena, Germany | 63x 1.4 NA oil objective | |
Neubauer improved cytometer | Labor Optik, Friedrichsdorf, Germany | 1100000 | Counting procedure: Wipe cytometer and coverslip provided with the counting chamber with 70 % ethanol. Press coverslip gently on the counting chamber so that the two glass surfaces are in contact and Newton's rings can be observed. Subsequently, 10 – 20 µl cell suspension can be applied to the edge of the coverslip to be sucked into the void by capillary action. Count the intact vs. defect myocytes using the squares of the cytometer grid which reflects 100 nl. Repeat counting procedure on the second grid provided on the cytometer. Calculate the density of cells in your original cell suspension by taking account of any dlutions and counting shortcuts. |
POC-R2 Imaging Chamber | Pecon, Erbach, Germany | Cell suspension volume: 800 µl; desired plating density: ~ 1000 AM and ~ 10,000 VM | |
Spring scissors, 8 mm blades straight, blunt | Fine Science Tools, Heidelberg, Germany | 15025-10 | |
Student dumont #7 forceps, inox | Fine Science Tools, Heidelberg, Germany | 91197-00 | |
Student iris scissors, curved, 11.5 cm | Fine Science Tools, Heidelberg, Germany | 91461-11 | |
Student iris scissors, straight, 11.5 cm | Fine Science Tools, Heidelberg, Germany | 91460-11 | |
Student surcigal scissors, straight, sharp, 12 cm | Fine Science Tools, Heidelberg, Germany | 91402-12 | |
Tissue forceps, 1×2 teeth, slim, 10 cm | Fine Science Tools, Heidelberg, Germany | 11023-10 |