Name: Assessment of EGFP-Positive Fiber Electrical Activity, Cysteine Staining, and Signal Acquisition
Uploaded: 2023-06-02T13:50:00
Description: Watch this Scientific Journal Video about Assessment of EGFP-Positive Fiber Electrical Activity, Cysteine Staining, and Signal Acquisition at JoVE.com

assessment of egfp-positive fiber electrical activity, cysteine staining, and signal acquisition

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Watch this Scientific Journal Video about Assessment of EGFP-Positive Fiber Electrical Activity, Cysteine Staining, and Signal Acquisition at JoVE.com

Assessment of EGFP-Positive Fiber Electrical Activity, Cysteine Staining, and Signal Acquisition  

Turn on all the acquisition setup components for the fluorometry measurement. Place the 35 millimeter glass bottom dish containing the dissociated muscle fibers on the stage of the microscope. Carefully remove the culture media with a 1, 000 microliter pipette, and replace it with 2 milliliters of room temperature ringer solution.Using a mechanical or motorized manipulator, place the two platinum wires perpendicular to the bottom of the dish. Ensure the electrode terminals are aligned relative to the fiber's longitudinal axis, and a few millimeters away from the fiber's ends. Adjust the electrode positioning by rotating the dish or moving each electrode if mounted on an independent micro manipulator.Turn on the transmitted light and find the fibers in the field of view using a 20 X objective. Move the EGFP filter cube into the light pathway. After turning off the transmitted light, activate the 488 nanometer excitation light using a remote controlled light shutter.To identify the EGFP positive fibers, center the fibers with the brightest EGFP signal in the middle of the field of view. Use a diaphragm positioned in the excitation light path to focus the excitation light in a specific area of the fiber. This enables signal acquisition, where the EGFP CaV1.1 signal is maximal.Adjust the fiber position with the mechanical stage to align with the diaphragm light path, and store the fiber XY location. After returning to the first saved localization, set the duration of two sequential stimulation pulses to 1 millisecond, amplitude to 20 volts, and the polarity of the pulses to alternating. Then, use a manual trigger switch to deliver the two sequential pulses.During the stimulation, observe two concentric homogenous fiber contractions in response to the two pulses of opposite polarity. If no contraction or only one concentric contraction is observed with alternating polarity pulses, discard the fibers from the rest of the experiment. Add 2 microliters of 10 millimolar MTS 5 TAMRA solution directly into the dish, and gently mix with a 1, 000 microliter pipette.Incubate for four to five minutes to allow the diffusion of the fluorescent thiol molecule into the transverse tubule system lumen. Through field stimulation, apply bipolar repetitive stimulations to evoke successive action potential trains at a rate of 50 hertz for 300 milliseconds every 1 second for 5 minutes. Remove the staining solution from the dish with a 1, 000 microliter pipette.And replace it with 2 milliliters of room temperature ringer solution. Let the stained fiber recover from the staining protocol for at least 10 minutes. After reassessing the fiber health and electrical activity with two alternating pulses, move the MTS 5 TAMRA filter cube into the light pathway, and activate the 533 nanometer excitation light with a remote controlled light shutter to visually confirm staining on the fibers.Next, using the stored location on the motorized stage, place the fiber in the middle of the field with the MTS 5 TAMRA filter cube, the diaphragm, and the 60X oil immersion objective. After reorienting the field stimulation platinum wires at each end of the fiber, align the wires on the main axis of the fibers in a straight line, and space them five millimeters apart, with the fiber in the center. On the acquisition software, select the protocol that will be used for acquisition.This step is controlled only with the computer, and triggers all the downstream devices for electrical stimulation and light path shutter control. For each protocol, minimize the total acquisition time as much as possible to avoid signal bleaching, but allow a few tens of milliseconds of light illumination and signal acquisition before the first electrical stimulation to record a baseline. Run the protocol by hitting Execute.The recorded signal is automatically displayed on the screen. The small amplitude of the signal and the quick mechanical contraction of the fiber often hide most of the signal, and display a movement artifact. Add 1 micromolar of 100 millimolar and benzoyl para toluene sulfonamide to the recording solution to minimize the contractual responses, and further distinguish the signal arising from S4 motions due to fiber activation.After a few minutes, run the same protocol a second time. The movement artifact is now removed, but a small fluorescence change corresponding to the S4 movement remains. Move the dish slightly using the mechanical stage to acquire a signal from an area without any fibers or debris.Run the protocol a final time to record the background fluorescence. Export the traces and use a spreadsheet program to express the signal as delta f over f 0 as a function of time, and apply baseline correction if required. The examples of transmitted and fluorescent images of the dissected and not dissociated muscle expressing an EGFP CaV1.1 construct are shown here.The representative images of a muscle fiber expressing an EGFP CaV1.1 VS D3 construct before and after MTS 5 TAMRA staining are shown in this figure. This dye also stains endogenous cysteines of non-transfected fibers. The confocal images of an EGFP CaV1.1 VS D3 construct and MTS 5 TAMRA staining show a double band pattern characteristic of CaV1.1 localization on the transverse tubule system of the muscle fiber.The representative fluorometric recording in response to two stimuli and measured with a photo diode is shown here. Both signals are normalized by their minimum value and plotted as a function of time relative to their respective depolarization. These recordings show that the rising phase and the time to peak of the signal are similar for both depolarizations imposed on the fiber.

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Biology

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability  

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