To calculate the precise [Ca2+]m μM the RLU obtained from the luminometer were introduced into the above equation. RLU denote the relative luminescence detected whilst DC denotes the dark count observed in a cell free system, (equalling 5 RLU).
6.9.2.
FlexStation 3
The FlexStation 3 (Molecular Devices) is an automated microplate reader that allowed for the real time measurement of fluorescence, whilst making automated experimental additions.
The fluorescence of the various dyes, Fura-2, Fluo-4, x-Rhod-1 and TMRE, was measured using the FlexStation 3. Cells were cultured in black walled 96 well plates, at a 4.0 x106 cell dilution. Once confluent, the cells were loaded with the required dye, as described in sections 6.2.1 and 6.7. The culture plate was then located in the FlexStation 3.
The reagents were five times concentrated, as they were added at a ratio of 1 in 5, to give the desired final concentrations. The reagents were used alone and in various combinations as required. Once prepared, the fluorescence was recorded using Softmax Pro software, Flex mode, allowing the addition of compounds alongside recordings. The programme setup is described in Figure 6-6 and the fluorescent settings in Figure 6-5.
The resulting fluorescence was plotted as F/F0, (fluorescence (F) divided by the average fluorescence during the base line reading from 0 to 300 seconds). This was
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then normalised to the control mock additions to eliminate any addition artefacts and to allow comparison between repeats.
6.9.3.
Tube Luminometer
Following incubation in the presence of the poison cocktail (as described in 6.8.1), a 2 μl sample of the buffer was collected. The sample was combined with 20 μl of ATP monitoring reagent (AMR, ViaLight®). The AMR emits light, relative to the ATP concentration. The emitted relative light units (RLU), was measured using a Berthold tube luminometer (LB955) and detected using a photon counter, with a 380 to 630nm spectral range. For optimal luciferase activity, the luminometer was maintained at an ambient temperature (18oC to 22oC).
Subsequent to the ATP RLU measurements, 10 μM phosphoenolpyruvate (PEP) and 10 units pyruvate kinase (PK), were added to the tubes. The enzymes converted ADP to ATP and the resulting luminescence recorded.
For each sample the mean bioluminescence was taken from the measurements and then converted from RLU into nucleotide concentration, using the calibration curves produced, as discussed below.
6.9.4.
Calibration curves
Calibration curves were constructed from serial ATP and ADP dilutions, 1 pM to 10 mM. The curves generated, were then used to convert the RLU to nucleotide concentrations.
The ATP calibration curve was plotted as, ATP concentration against RLU (Figure 8-1). The ADP calibration curve plotted ADP standards against ATP equivalence (Figure 8-1). The ATP equivalence was calculated from the RLU generated from ADP converted to ATP minus the original count. Both curves were described by a Boltzmann curve of best fit, due to their sigmoidal shape and from this the nucleotide concentration was calculated, Equation 6-3.
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6.9.5.
High Performance Liquid Chromatography
Nucleotide species were separated and quantified using High Performance Liquid Chromatography (HPLC; Shimadzu), in parallel to the tube luminometer. The protocol used, is based on the based that published by Lazarowski et al. [319]. A 50 μl sample of buffer solution, from cells subjected to poisoning, as described in section 6.8.1, was collected. The buffer sample was heat treated at 100oC, for 10 minutes, using a heat block Thermo Cycler (PTC-200, Peltier). Heat treating the samples, inhibited any subsequent nucleotide interconversion. The samples were then treated with 10% Perchloric Acid (5.8M) for 30 seconds, at 22oC. The samples were then centrifuged at 1500x g, at 4oC, for 10 minutes. The supernatant was then collected ready for nucleotide sampling.
The treated samples were transferred into HPLC vials and loaded in the auto- sampler. The ASI-100 automated sample injector (Dionex), sequentially ran the samples through a Hypersil BDS C18 column (5 μm, 150x4.6mm). The ion pairing mobile phase was developed at 1 ml/minute from: 0 to 4 minutes in 100% solvent A and 4 to 30 minutes in 100% solvent B. The solvents were added using a P680 HPLC pump (Dionex). The nucleotides ATP and ADP were eluted at 23 minutes and 14 minutes, respectively [319, 614]. The absorbance was measured at 254 nm and monitored on-line, using a Dionex UVD 170U detector.
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6.10.
Confocal Microscopy
Images were acquired using an inverted Leica SP2 AOBS, 2 -photon laser scanning confocal microscope.
Live cell imaging was recorded using the confocal microscope. The cells were cultured on glass bottom 35mm petri dishes, as described in section 6.5.1- Calcium Indicators. Once loaded with the Ca2+-sensitive indicators, culture dishes were placed on the microscope stage and the cells located within the plane of focus. Live fluorescence was recorded in ‘real time’ capture mode at the relevant fluorescence as described in Figure 6-5. at described laser lines and emission bands or where to find them.
6.10.1.Immunofluorescence
Immunofluorescence was used to label cell structures in both cultured cells and cryostat sections, using the antibodies described in Figure 6-7 and Figure 6-8.
Prior to immunofluorescence staining, cells were fixed using PFA. Cells were cultured on 16mm diameter round glass coverslips at 2.0 x 106 cells. Once confluent the cells were washed, 3 times in PBS. The coverslips were then bathed for 15 minutes, in 1 ml of 4% PFA/PBS (v/v), at 22oC. The PFA was replaced with, 1 ml 50 mM ammonium chloride (NH4Cl), for 20 minutes. The ammonium chloride reduces potential auto-fluorescence, quenching free aldehydes [615]. The fixed cells were permeabilised with 0.2% TritonX-100, for 4 minutes. The coverslips were washed 3 times, for 2 minute in PBS.
Once permeabilised, the cells were blocked in a 10% v/v, serum/PBS solution and incubated for 30 minutes, at 22oC, in the absence of light. The serum was specific to the host of the secondary antibody. This was replaced with a 5% serum/PBS solution, containing the primary antibody(s) and incubated for 30 minutes, at 22oC, in the absence of light. The coverslips were washed, 3 times in PBS, for 4 minutes. Following this they were incubated in 5% serum/PBS, with the desired secondary antibody(s), for a further 30 minutes, as before.
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When the primary antibodies were generated from the same host species, direct staining was used. In direct staining, the primary and secondary antibodies were incubated in succession. If two structures were labelled the staining protocol was repeated after the initial staining. Where the antibodies were generated from different host species, indirect staining was used. This method involved co- incubation of the primary antibodies, followed by the secondary antibodies.
Once stained the coverslips were subject to a further 3 PBS, 4 minute washes. The coverslips were then immersed in PBS and twice in d.H20. Excess water was removed using filter paper. The coverslips, were mounted on 26 mm by 76 mm superfrosted slides using, 15 μl moviol with 4',6-Diamidino-2-phenylindole (DAPI, 1/2000).
Fixed and labelled cells were located on the confocal microscope stage and imaged using the 63 time oil immersion objective. The cells were captured using sequential imaging at 1024 by 1024 format. Sequential mode removed any bleeding between the fluorescence emission channels, ensuring there was no interference between labels.
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Dye Incubation Time De-esterify Time Temperature
Fura-2 (2.5 μM) 45 minutes 15 minutes 37oC
Fluo-4 (2 μM) 45 minutes 30 minutes 22oC
x-Rhod-1 (1 μM) 60 minutes 30 minutes 37oC
Figure 6-4. Table outlining the protocol for calcium dyes.
The table records the varying concentration levels and incubation times and conditions of the 3 different calcium sensitive fluorescent indicators utilised.
Dye Excitation Emission Cut Off
Fura-2 340/380 nm 510 nm 455 nm
Fluo-4 488 nm 530 nm 515 nm
x-Rhod-1 540 nm 600 nm 570 nm
TMRE 549 nm 574 nm 570 nm
Figure 6-5. Table of dye excitation and emission.
The table shows the excitation and emission of the dyes used with the FlexStation 3 and the cut-off.
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Figure 6-6. Overview of the FlexStation 3 software, Softmax Pro, settings.
FlexStation 3, SoftMax Pro, Flex mode, programme was set with the appropriate, excitation and emission wavelength. The Sensitivity was set to readings: 6 and PMT: medium, whilst Timing establishes the total run time, Time: 1500s and Interval: minimal interval. The Automix and AutoCalibrate were left off, while the Assay Plate Type was set as 96 Well Standard and Wells To Read set dependant on how many columns were cultured. The Compound Source left as “Beckman 140504” and Compound Transfer set to 2 Transfers; transfer 1: pipette height = 200 μl, volume = 25 μl, rate = 2 and time point 300secs and transfer 2: pipette height = 200 μl, volume = 30 μl, rate = 2 and time point 1500secs. Triturate was not used, Pipette Tips Layout full rack, Compound & Tip Columns set so the tips column and compound fill are the same for each column and AutoRead off.
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Primary Antibodies Host Species Notes
anti-Troponin T Rabbit Cardiac isoform Ab-1 (clone 13-11) [616]. (Thermo Scientific MS-295-P0)
anti-sarcomeric α-Actinin Mouse Derived from clone EA-53.
(Sigma A7811)
anti-myosin heavy chain Mouse Sarcomeric specific [617, 618]
(DSHB MF-20)
Figure 6-7. Table of primary antibodies for immunofluorescence labelling.
The primary antibodies used and their respective host species and any additional information regarding the generation.
Host Species Reactivity Label or Dye Excitation/ Emission
Goat anti-rabbit* Alexa Fluor® 488
(A-11012) 495/519 nm
Goat anti-mouse* Alexa Fluor® 488
(A-11001) 495/519 nm
Goat anti-mouse* Alexa Fluor® 594
(A-11008) 590/617 nm
Figure 6-8. Table of secondary antibodies for immunofluorescence labelling. Thesecondary antibodies bind to the primary antibody, depending on their reactivity. They are labelled with an Alexa Flour® green or red tag, detectable at the set excitation and emission values. *All reactivity was IgG target isotype.
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6.11.
Statistics and Data Presentation
6.11.1.Plotted Data
In several circumstance the average from multiple and comparable experiments was plotted to represent the observed results. In several circumstances a line of best fit was applied to the data, including the Boltzmann Curve as described below.