Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.
The over all design of the protocol containing four major steps is depicted in Figure 1. All steps are described in full detail. Cell isolation, transduction and culture, followed by confocal imaging about 24 hours later is a consecutive and compulsory timescale. The virus construction should be done ahead of the cell isolation and is then used for the genetic transduction.
1. Isolation of adult cardiac myocytes from rat heart
2. Construction of an adenovirus for fusion protein transduction
To produce adenoviruses the Transpose-Ad Adenoviral Vector System from MP Biomedicals is used1. This protocol starts with the available and tested entry vector (pCR259) for a fluorescent fusion protein or GEB s2.
3. Primary cell culture and adenoviral transduction
4. Confocal imaging for 3D subcellular structures and Ca2+ Signaling
Confocal imaging can be performed on any confocal microscope. However, calcium-imaging experiments require an acquisition speed of at least video rate (25-30 Hz). This restricts usable technologies to either multi beam scanners (e.g. Nipkow disc, swept field or kilo-beam array scanner) or the very fast single beam scanner (e.g. resonant scanner or acousto optical deflector (AOD)-scanner). For an overview see Ref. 3. The 3D-subcellular imaging requires a z-drive. Here a kilo-beam array scanner (VTinfinity, VisiTech Int., Sunderland, UK) is used.
5. Representative Results:
The final outcomes of the protocol are image sequences that can be used to extract further data.
An example is the investigation of subcellular structures and organelles. Figure 3 depicts the example of the 3-dimensional arrangement of the Golgi apparatus. In contrast to electron microscopy all investigations can be performed on living cells.
Figure 1: General flow of the major experimental steps.
Figure 2: The left image depicts the Q-HEK cells in culture with 90-95% confluency which are not transduced. Cytopatic effects after viral transduction are shown in the right part. Cells round up and detach from the bottom of the plate. In these cells the nucleus occupies the major part of the cells as a result of active virus production.
Figure 3: Adult rat cardiac myocyte transfected with an YFP-fusion protein targeting the Golgi apparatus. Cardiac myocytes after adenoviral gene transfer (one day in culture) express the fluorescent fusion protein. Panel A depicts a single confocal slice taken from a z-stack. In panel B the same cell is depicted after deconvolution using a theoretical point spread function (PSF). This de-blurred image stack is subsequently used to render the cell and get a 3D volume reconstruction as shown in panel C or a 3D surface reconstruction (D). The bar represents 10 μm.
The procedure described for the isolation of rat cardiomyocytes can be adapted to other species, e.g. mouse4, if necessary. A parameter that needs adaptation is the enzyme mix for digestion (see below) and also the duration of digestion. Be aware that the cells of some species (e.g. mouse) might be more fragile than others (e.g. rat).
The choice of collagenase is probably the most crucial step in the isolation procedure. We choose Liberase Blendzyme because it is a synthetic enzymatic mix with virtually no batch-to-batch variations. The conventional method is the use of so called crude collagenase extracted from Clostridium histolyticum, which is also a valid method. However, batch-to-batch variations require new optimizing for each new batch.
Apart from the fluorescent fusion proteins and the GEB transduction the adenoviral gene transfer can also be used for over expression of functional proteins or their down regulation (e.g. by RNAi5). This holds true because transduction efficiency is high (> 95%) and reproducible.
This work was supported by the German National Science Foundation (DFG) and by the Federal Institute for Risk Assessment (BfR, Germany).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Anesthesia solution | Serumwerk Bernburg GmbH (Ursotamin), Bayer Health Care (Rompun) | Mixture of 1 ml Ursotamin (100 mg/ml Ketaminhydrochlorid) and 240 μl Rompun (2% Xylazinhydrochlorid) | ||
Citrate solution | 117,64 mg sodium citrate in 10 ml 0.9% NaCl solution | |||
Solution A | 134 mM NaCl, 4 mM KCl, 11 mM glucose, 1.2 mM MgSO4, 1.2 mM Na2HPO4, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered | |||
Solution A+ | Content of solution A plus 0.2 mM EGTA, pH adjusted to 7.35 using 10 M NaOH, sterile filtered | |||
Liberase solution | F. Hoffmann‐ La Roche Ltd. | 11988476 001 | 500 μl stock solution in 15 ml solution A (stock solution: 10 mg/ml Liberase Blendzyme 4 in aqua dest.) | |
Solution B | Content of solution A plus 200 μM CaCl2 and 0.1% DNase solution | |||
Solution B1/2 | 1:1 mixture of solution A and solution B | |||
DNase solution | Sigma‐ Aldrich Co. | D4527 | Deoxyribonuclease I, Type 2 from bovine pancreas, 40 KU (15 mg) are dissolved in 5 ml of 10 mM Tris‐HCl buffer, adjusted to pH=7.35, additional content: 50 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol; this solution is mixed with 5 ml glycerol | |
Extracellular matrix protein (ECM) solution | Extracellular matrix protein (ECM) solution | E1270 | 1 ml stock solution as purchased is diluted with 6 mL medium M199 | |
Culture medium M199 | PAA | E15‐834 | Medium M199 supplemented with 1 μl/ml ITS solution and 20 μl/ml penicillin‐streptomycin solution (5000 units/ml) | |
ITS solution | 25 mg Insulin, 25 mg Transferrin and 50 μl Selenite stock solution are dissolved in 5 ml aqua dest. (stock solution: 0.5 mg/ml Sodiumselenite in aqua dest.). Add a few drops of 1 M HCl until one should get a clear solution. Aliquots can be frozen. | |||
Tyrode | 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 2mM MgCl2, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered | |||
Master mix | 5 μl 10×PCR‐Buffer, 5 μl MgCl2 (25 mM), 2 μl forward primer, 2 μl reverse primer, 1 μl dNTP´s (10 mM each), 33 μl H2O, 1 μl DMSO, 1 μl Taq‐DNA polymerase for each sample | |||
LB‐Medium | 10 g/l Tryptone, 5 g/l Yeast extract, 5 g/l NaCl (and 15 g/l Agar if used as solid medium), pH 7.0 | |||
Pac I | New England Biolabs | R0547L | preparation: 50 μg DNA, 5 μl 10×buffer, 0.5 μl 100×BSA, 0.75 μl Pac I, fill with H2O up to final volume of 50μl |