The best principle of the Fluorescence correlation spectroscopy, FCS, was invented initially in the 1970s. In the 1990s, important and many improvements for FCS was established by combining with a confocal apparatus microscopy. Since then, FCS has been used for many chemical and virus cure applications, such as molecular interactions and protein aggregation analysis.
Protein aggregation is a hallmark of neurodegenerative disorders. Fluorescence brightness are single particles in action of molecular sizes explained by diffusion properties. It's very important in measuring protein aggregations because it can determine projected life cycles of molecules, but also distinguish the homo or hetero polymerization.
Here, we introduce a procedure to measure diffusion of amyotrophic lateral sclerosis associated with aggregated form protein using FCS in cell lysates and live cells. Prepare 100 millimeter plastic dish growing Neuro2a cell sitting comfortably in the normal growth medium. Confirm the cell confluency.
Remove the medium. Add 3.5 milliliters of Trypsin-EDTA solution into the third cartridge dish and incubate them at 37 degrees celsius for one minute. Add 9.5 milliliter normal growth medium into the dish and suspend them.
The cell suspension is mixed with trypan blue to stain the dead cells. Add suspension to the cell-counting slide. Count the number of cells using the cell counter, or manually.
Check the live cell number and their viability. Dilute the cell in the counted medium at 1.0 times 10 to the power of 5 per milliliter. Add two milliliters of cell suspension into the 35 millimeter plastic dish for cell lysis or growth space dish for live cell measurement.
Incubate the dish at 37 degrees celsius for one day. The next day, prepare the mixture of plasmid DNA and the transfection reagents. Add the transfection mixture to the cell-counted medium and incubate the cells for 24 hours.
One day later. Check the GFP expression using a routine microscope. You can see the very bright TDP25 ingredient bodies in cells.
Cell lysis should be performed at the biochemical bench. Remove the medium in the dish. Add two milliliters of PBS at 25 degrees celsius to wash as a medium.
Remove the PBS. Place the dish on an aluminum plate on the top of the crushed ice. Immediately, add the 200 microliter lysis buffer into the dish.
Scrape the dish using a cell scraper. Recover the lysate with undissolved cell debris in a new 1.5 millimeter tube. Centrifuge the lysate at four degrees celsius.
For live cell measurements, replace the medium with a fresh one before the measurement. Check the cell attachment using a phase-contrast microscope. Also, check the GFP expression using a fluorescence microscope.
Use a confocal microscope-combined FCS system. Turn on the 488 nanometer laser. Stabilize the system, at least, for 30 minutes.
Set up the optical path. Use a 488 nanometer laser for the instant light. Use an appropriate beam splitter and dichroic meters.
And also, fluorescence fusers. Usually, we'll use coverglass chambers for calibration and solution measurements. Carefully, take the coverglass chamber.
Place the glass surface on the lens screening paper. Pour the FCS calibration. Add the Rhodamine 6G solution in as well.
Add the ultrapure water on the objective. Do not use the oil. Set the chamber on the microscope stage.
Move the stage to the appropriate position. Create the pinhole adjustment and open the pinhole adjustment wizard. Monitor the photon count rate as coarse movement of the pinhole for the x-direction.
The brightest position was rightly determined. Next, monitor the photon count rate as fine movement. The brightest position was successfully determined This is the pinhole position for x-direction.
As the same way, search the brightest position of the pinhole for y-direction. The brightest position for y-direction was also successfully determined. Compound the x and y position of the pinhole.
It is better to adjust the pinhole position before each day's measurement. When the live path is changed, the pinhole position must be adjusted again. Create a count rate and monitor the phone count rate.
Change to the CPM monitoring mode. Count the correction ring of the object you ran so that the CPM value is the highest. Close the count rate window.
Start with the Rhodamine 6G solution measurement. After the measurement is completed, click the fit to perform Curve Fitting Analysis. Select a model for fitting of the old correlation function.
For Rhodamine 6G, select the model for 1-component, 3-dimensional diffusion with a triplet state. Set the fitting start time by moving the red line. Click Fit All, to start the fitting calculation.
Check the fit deviation. Make sure that sides are posted and negative, around zero. Check the fitted values.
Make sure the diffusion time is roughly in the range of from 20-30 microseconds. Moreover, structural parameter is from four to eight. Open the fit panel, and select a model for the measurement.
Enter the same structural parameter value in the model for the sample in the fit panel. Make sure the type should be set as Fixed. GFP-TDP25 measurement in cell lysate.
Place the lysate in the coverglass chamber. Place the lid to avoid dry up of the lysate. Place the stage lid for light shading.
In the acquisition panel, set the laser power, measurement time, and its repetitions. Start the acquisition. A spike was observed.
A spike was observed again. The spike indicates bright molecules passed through the detection body. Spikes indicate soluble oligomers or aggregates.
Create a fit to perform curve fitting analysis. Select the model for 2-component, 3-dimensional diffusion with triplet state. Set the fitting start time.
Click the Fit All. Check the fitted values. SOD1-G85R tagged with GFP measurement in a live cell.
Unlike solution measurement, I recommend using the heat stage incubator. Set the cell-culturing dish on the microscope stage. Confirm the focus and the position using the ocular.
Select a measuring cell. Zoom-in and adjust the cell position using a fast scanning mode. Acquire the snapshot of the cells using the slow scanning speed mode.
Select an FCS-measuring position using the Position tool. The crosshair indicates the measuring position. Start the measurement.
The slight decrease of the photon count rate record suggests photobleaching of GFP. Perform the curve fitting. Representative results of GFP-TDP25 in cell lysate is shown.
The top graph a record of photon count rate. Where there's arrows is a spike, suggesting soluble oligomers and aggregates. The middle graph represents the old correlation function.
The gray line shows the low, old correlation function. The magenta line shows the fitted function. The dotted lines indicate the start and end times for fitting.
The bottom table shows the fitted value. Next, representative results of SOD1-G85R-GFP in a live cell are shown. The top graph represents a record of our photon count rate.
The gradual decrease of the recorded photon count rate was observed, suggesting photobleaching of GFP growth for different measurements. This is because of the slow diffusion speed in live cells. The middle graph represents the old correlation function of SOD1.
The gray line shows the low, old correlation function. The magenta line show the fitted function. The dotted lines indicate the start and end times with fitting.
The bottom table shows fitted values. Here we show you this procedure to measure diffusion from every cell associated TDP-25 in cell lysate and everything mutant of SOD1 in live cells using FCS. The soluble oligomers and aggregation in cell lysate can be often observed as spikes or bursts.
On the other hand, in live cells, such spikes are rarely observed, except for possibly obvious live structures. A slowly diffusing species of mutant SOD1's add mean brightness for single particles such as SOD1 homopolymerization inside for us. The procedure shown here is simple and worthwhile.
FCS contains no radiation;directly dormant state. It's just your, and our, imagination empathy.
