This experimental method is used to reveal the properties of store operated calcium entry or SOCE in cancer and skeletal muscle cells using fluorescence based methods. This is achieved by recording the fluorescence of URA two in live cells, which is sensitive to intracellular calcium concentration. In addition, the fluorescence quenching rate by addition of manganese at calcium concentration insensitive wavelength is measured, which directly reflects the activation of SOCE to study SOCE.
In structurally specialized skeletal muscle cells, muscles are dissected and skinned. Muscle fibers are prepared. The fibers are then treated with low affinity calcium indicators and their internal fluorescence is monitored.
These methods demonstrate that SOCE exists in both non excitable cells and skeletal muscles with graded activation and tight coupling to intracellular calcium store content. The main advantage of this fluorescein based technique over other methods like electrophysiology, patch clan, is that it is convenient to use and can be applied to cell population study or high throughput screening. It Is important to use the right esthetician S for ritual metric environment and mag coning assay.
So calibration of individual instrument is crucial because each instrument is different. Visual demonstration of this method is critical as the mechanical skin process of single muscle fibers is difficult to learn because it requires a special hand-on training indepth understanding and learning of micro dissection techniques, careful observation, and a really good dose of patients Before beginning, calibrate the microscope setup for measurement of URA two fluorescence. Although the excitation wavelengths for calcium binding and calcium three URA two are three 40 and the three 80 nanometer, the best ratio dynamic range of URA two for calcium measurement may occur at other wavelengths in a particular microscope system.
Such shifts in wavelengths are due to changes in the optical path with the additional various optical components. Similarly, the ISO spastic point for each system varies too. Therefore, it is important to determine this wavelengths by spectrum scanning.
Alternatively, Ming screening can be recorded by a just fluorescent at any two wavelengths in such a way that the final value is independent of calcium concentration. Begin by transecting cultured K Ys SE one 50 cells with plasmids expressing RFP and containing either HRNA specifically against or I one or a scrambled sequence for 48 hours. Grow the transfected cells on glass bottom dishes.
Then remove the medium and rinse the cells with a balanced salt solution or BSS. After the rinse, add one milliliter of BSS containing two micromolar RA 2:00 AM and keep the dish at 37 degrees Celsius for 40 minutes. Put the cells at room temperature for 15 minutes to allow intracellular URA 2:00 AM form to convert into URA two.
Then wash the plate twice with BSS under the microscope. In visualization mode, select cells expressing RFP. Select the excitation ratio mode with the emission wavelength set to 510 nanometers and the excitation wavelengths at 350 and 390 nanometers.
The ratio is displayed in the third window. Next, record. The selected cells fluorescence at the predetermined wavelengths while the cells are perfused with four different solutions.
Similarly, KYSE one 50 cells are loaded with ferra two and subject to recording. In excitation ratio mode, unlike the previous procedure, the ISO spastic point 360 nanometers is chosen as an excitation wavelength. Then simultaneously record the fluorescence at 360 and 390 nanometers while the cells are perfumed with five different BSS solutions.
For this protocol, C two C 12 cells, a mouse myogenic cell line must be cultured on glass bottom dishes to confluence, and then differentiated into myo tubes in differentiation medium containing 2.5%horse serum. Load the C two C 12 myo tubes with a final concentration of five micromolar RA 2:00 AM in BSS solution and incubate them for 40 minutes. Then add a final concentration of 20 micro molars and benzo p toluene smide or BTS to inhibit muscle contractions and the motion artifacts caused by them.
Incubate the cells for 15 minutes at room temperature and remain BTS in all solutions. Next load four. Balance salt solutions into the perfusion system.
As previously described. During each profusion record the fluorescence of selected cells at 360 and 390 nanometers. In this series, magnesium and thap argen are applied together to monitor magnesium ion quenching.
While endoplasmic reticulum calcium stores are depleting. To dissect the extensor digitorum longus or EDL muscle, position the leg of a euthanized mouse laterally and remove the skin from the ankle area up to the knee. Cut the superficial muscle layers of tissue to expose the EDL and sever both the upper and lower tendons to free the intact EDL muscle.
It is important to keep the length of tendons as long as possible. Now to prevent any contractions, transfer the EDL to a T road solution containing 0.1 millimolar EGTA and no calcium under a stereo microscope. Force the knee insertion tendons into two halves and split the EDL muscle into two bundles.
Repeat the process until eight bundles are obtained. If tendons are lost from any bundle, discard the bundle. Next, grip both tendons of an EDL bundle strip and carefully stretch the strip until three or four separated Single muscle fibers are left intact with tendon tissue on both sides.
Put a drop of calcium free tyro buffer in a glass bottom dish. Lay the EDL strips straight on the dish and quickly remove as much solution as possible. Next, tightly tape down both tendons using water resistant scotch tape.
Wash the fiber first with the calcium free solution and next twice with a 2.5 millimolar calcium solution. If the fiber hyper contracts and damages noticed, discard the fiber, wash the selected healthy muscle fibers three times with wash solution number one, and then bathe them in modified intracellular like skinning solution for five minutes. Using a tungsten needle attached to a pin holder mechanically skin the fiber under a stereo microscope, the fibers will reseal and trap road five n conjugated with calcium in the transverse tubules.
Obtaining health single intact muscle fibers or at least bundles of these types of fibers that still have some tendon attached to them is, in my opinion, one of the most challenging steps followed by the always very difficult transfer of these fibers to the final experimental chamber. During this process, fibers might hyper contract or they might even break during this process. So if you have healthy fibers, the mechanical is skin itself is going to be much easier To load the SR.Incubate the fibers and skinning solution with 20 micromolar flow five N am.
Follow the incubation with extensive washes and washing solution number two and 30 minutes of additional incubation to allow complete de esterification of the dye. The fibers can then be visualized using a confocal microscope with a 60 x objective from regions of interest. Simultaneously record the fluorescence of each dye while the fiber is perfused with SR loading SR Depletion solutions.
SOCE activity in KYS, SE one 50 cells was examined using the intracellular calcium measurement. The cells were code transected with RFP and a short hairpin, RNA against the OR I one gene, which encodes A-S-O-C-E channel compared to controls represented by the black trace knocking down or I one expression resulted in decreased SOCE activity, SOCE and KYC one 50 cells was also confirmed with the manganese quenching assay. After TG completely depleted ER calcium stores, the profusion of manganese resulted in a significant fluorescence decrease.
The steeper slope of URA two signal loss indicates a more active SOCE. Thus, the slow fluorescence decrease in two A PB treated cells indicates that SOCE activity was blocked by this compound. The manganese quenching rates were determined from the initial 10 seconds where the quenching was still in linear range without saturation, a similar manganese quenching assay was performed in cultured muscle cells.
In this case, manganese was applied together with tg. While TG was depleting the SR calcium stores, the fluorescent quenching slope gradually changed until it reached its maximal rate. The sigmoidal curve indicated the graded activation of SOCE under these experimental conditions.
Under confocal imaging skinned fiber trapping of road five and die in the TT compartment showed the characteristic mammalian doublet pattern as seen in the red channel. After loading of the SR with flow five NM, the typical punctuated SR pattern was visible in the green channel upon profusion of the skinned fiber with TTSR loading solution fluorescence intensity of both road five N and flow five n increased upon profusion with SR depletion solution fluorescence levels in the SR compartment and the TT compartment started to decrease. This indicated that there was coupling between SR calcium store depletion and SOCE activation respectively.
After watching this video, you should have a really good understanding of how to obtain first viable really healthy, intact muscle fibers. They, they need to have enough tendon at their ends to allow you an effective skin process, which ultimately will allow the experimenter to have a physiological relevant preparation that has an incredible advantage. Don't forget that all fluorescent styles used in this study are usually light sensitive and precautions, such as loading in the dark dish and the washing in the dark room should always be practiced while performing this procedure.
O mastered this technique can be done in a few hours. What we suggest is that several intact fibers are first obtained to allow for a higher percentage of success with the mechanical skinny steps, and also if you want to culture these fibers overnight After its development. This technique paved the way to researchers in the field of calcium signaling to explore the role of store operated calcium entry in the growth and death of cancer cells.
The physiological and pathological function in muscle.
