The overall goal of this protocol is to measure the diffusion rate of a labeled protein in membranes of primary immune cells using fluorescence correlation spectroscopy. This method can help identify key questions in the immunology and cell biology fields, such as identifying natural killer cells with a more active membrane receptor dynamics. The main advantage of this technique is that it is used with live cells, so it accurately reflects the natural state on how molecules move.
The implications of this technique extend towards immunotherapy of cancer if different natural killer cell traits, such as molecular diffusion, are linked to their function that efficiency can be predicted better. Though this method can provide insight into molecular movement in immune cells, it can also be applied to other primary cells and cell lines in any discipline. Open the software in expert mode, with the option Scan New Images selected.
Select the 40X water objective lens on the confocal laser scanning microscope. Under the Acquire tab, press the Config and Scan buttons to open the Configuration Control and the Scan Control. Under the ConfoCor tab, press Measure to open the Measurement window.
Next, create a folder to save the FCS measurements. Start a new image database by clicking New under the File tab. Turn on the halogen lamp.
Using the Laser button, turn on the relevant lasers for exciting the fluorophores. Then specify the settings for image capture in the Configuration Control window. This includes the excitation laser, excitation and emission filters, light path, and detectors in use.
Similarly, go to the Measurement window and adjust the FCS settings under the System Configuration tab. Activate the detection channels for the FCS recordings by toggling Ch 1 for the green channel, and Ch 2 for the red channel. While the laser stabilizes, which may take up to 30 minutes, adjust the pinhole.
First, place a 50 microliter drop of dye centrally into one well of an eight-well chambered coverglass slide coated with poly-L-lysine. The adjacent wells are used to make cell measurements as the calibration is slide-dependent. Put a drop of distilled water on the 40X water objective lens and position the slide.
Then press Vis to select the visible light source and focus on the central position of the fluorophore droplet. In the software Measurement window, select the Acquisition tab and under Positions, toggle the current position. Then return to the System Configuration tab in the same window, and set a high or saturating power for the green laser.
To test the stability of the signal, press Start to open a new window displaying the Time trace and Auto correlation curve. Check that the fluorescent signal is high, stable, and that the y axis shows fluctuation in the kilohertz range. Lower values could be due to imprecise focusing or dye degradation.
Next, in the Measurement window, select the O Adjust button. Then select the correct detection channel and press Auto Adjust X.If the upper limit of the resulting curve is missing or not centered, mark the Coarse option and press Auto Adjust X again. After adjusting the X direction, do the same for the Y direction by pressing on Auto Adjust Y.Deselect Coarse and alternate between adjusting X and Y by pressing Auto Adjust until both have clear maxima and stable values.
If the cells labeled by two different fluorophores, add a drop of red fluorophore solution to an adjacent chamber. Then calibrate the second channel. Set the red laser to a high power, and repeat the stability calibration procedure.
Next, we measure the transit time of freely-diffusing fluorophores through the focus. And we use the stage then to calculate the area of the cell membrane that is within the focus. To start, focus on a droplet of solution by carefully carrying out the adjustment procedure.
Then go to the Acquisition tab in the Measurement window and set the acquisition time to 30 seconds with three repeats. Set the laser power at near saturating and press Start to take a measurement. Record the counts per molecule or CPM after the measurement is complete.
Then take new measurements making iterative reductions to the laser power. Within this power range, include the expected power of the forthcoming cell measurements. Next check that the CPM values scale linearly with the laser power.
Provided the rest of the scale is linear, saturation at the highest power is acceptable. The first time a newly-conjugated fluorescent antibody is used, perform an FCS measurement of freely-diffusing antibodies to quantify the expected CPM. And it is important to use wells coated with PEG Poly-L-Lysine for free antibodies.
For this procedure, coat a chambered slide with 200 microliters of Poly-L-Lysine grafted to polyethylene glycol. Let the solution adhere for an hour at room temperature. The stock solution can be refrigerated for up to two weeks.
And the powder stock should be stored in the freezer. Later, remove the liquid and allow the chamber to air dry. At the microscope, dilute the antibody in PBS to between one and 10 micrograms per milliliter.
Proceed to measure the transit time of free antibodies as described in the previous section for free fluorophores. To take measurement in a cell, add 125 microliters of cells in PBS plus 1%FCS from the antibody-labeled cell suspension. And 150 microliters of transparent Roswell Park Memorial Institute Medium 1640 into an empty well in the PLL-coated glass slide.
Let the cells incubate in the dark at the measurement temperature for at least 20 minutes before proceeding, so that the cells can attach and equilibrate. Start the imaging with fast XY scans to find the lowest possible laser power needed to view the cells. Then center the image on a round cell of average brightness where the membrane is clearly defined.
It is crucial that there is no fluorescent signal in the cytoplasm. Zoom in until the cell covers most of the image. Then focus on the top of the cell membrane.
Start at the center of the cell and move upwards until the center of the image shows a small round area with even fluorescence. Careful placement of the focus is crucial. If the focus is not in the membrane, the fluorescence detected will not come from the fluorescently-labeled membrane receptors.
And thus the results will not be reflective of their behavior. Now under the Acquisition tab of the Measurement window, select a position for FCS measurement by activating the Crosshair. Then set the number of repeats to between six and 10 and set the Measure Time to 10 seconds per repeat.
Now return to the System Configuration tab of the Measurement window and lower the laser power for FCS measurements to between one and 10 microwatts. Then press Start. If the intensity trace drops by more than 30%during the first 10 seconds, the cell has then been bleached too much and another cell must be measured at a lower power.
If the signal is lost, adjust the focus in the Z direction and restart the measurement. After the measurement is complete look in the Scan Control window to check that the cell is still centered to be sure that its membrane was measured. If the cell moved, discard the data.
Now go to the Auto Correlation window and manually delete individual repeats that contain zooming maneuvers, bleaching, large clusters, or other artifacts. Use a right click and select Delete to remove the artifacts. Selecting the actual artifacts requires practice.
The removal of repeats is a simple process, but the repeats need to be assessed correctly to know what should be removed and which should not. Then save the final file in FCS format. Keep only cells that have at least four repeats left after the removal of artifacts for further analysis.
A typical membrane receptor Auto correlation curve is smooth and has a transit time in the range of 10 to 400 milliseconds. Set the fitting domains so that both the start and the end of the steepest sloping portion, representing diffusion, is represented. Using a model of free diffusion in two dimensions to fit the data, pay attention to the most deeply-sloped portion of the curve.
Small fluctuations at or around one second at the tau scale, seen in the residual, are acceptable. If the curve fits badly, but the cell has no apparent problems, such as moving, bleaching, or clusters, then the starting values or upper and lower limits of the model likely need changing. Once the model fits well to the Auto correlation curve, extract the values of the variable parameters.
The number of molecules can vary between 0.5 to around 200 per square micron for endogenously-expressed proteins. Check that the CPM value from the cell measurement is at least 33%of that recorded from freely diffusing antibodies. After watching this video, you should have a good understanding of how to measure the diffusion rate of proteins using fluorescent correlation spectroscopy.
Once mastered, this technique can be performed in five to six hours if performed properly. While attempting this procedure, it is important to keep the cells on ice until around 20 minutes before the start of measurement. Do not measure any samples more than two hours.
Following this procedure, other methods like imaging can be performed in order to answer additional questions, such as how the diffusion rate correlates to the expression level of a certain protein. After its development, this technique paved the way for researchers in the fields of molecular biology and immunology to explore dynamics of proteins in live cells.
