Fluorescence cross-correlation spectroscopy is a statistical method that uses time result fluorescence to pinpoint the dynamic signature of G protein-coupled receptors in living cells. Here, we are particularly interested, in beta-2 adrenergic receptors. Sphingolipid receptors mainly provide information about translational diffusion dynamics and with the help of fluorescence anisotropy also rotational diffusion.
By introducing an additional label, we can also pro-binding or even conformational changes if foster resonance energy transfer between the labels as probed. Quantitative time resolved fluorescence requires careful alignment of the setup and calibration measurements. In the following minutes, we will provide an experimental guide for life cell fluorescence correlation spectroscopy, cross correlation spectroscopy, and Forster resonance energy transfer of G protein-coupled receptors, using clone able tags and synthetic flow force.
Cell sitting and transfixing need to be performed under sterile conditions. Place a clean cover slip barbell onto a six well culture plate, and wash it down with sterile phosphate buffer saline add two mL of cell culture medium with phenol red supplemented with 10%fetal bovine serum, 100 microgram, per amine penicillin and 100 microgram per ml streptomycin to each well, and keep it aside. Take the CHO cells which are cultured in the same medium with phenol red at 37 degrees centigrade in five percent CO2 and wash them with five mL PBS to remove the dead cells.
Add two mL of trypsin and incubate for two minutes at room temperature. Dilute the data cells with eight mL of medium with phenol red and mix carefully by pipetting. Count the cells in a Neubauer chamber and sit the cells at a density of 150, 000 cells per well in the six well culture plate containing the cover slip.
Let the cells grow in an incubator for 24 hours in order to achieve approximate 80%confluency. Dilute two microgram of the desired vector DNA. For example, CT SNAP or NT SNAP, and six microliter of that transfection reagent into two separate tubes.
Each containing 500 microliter reduced serum medium for each well and incubate them for five minutes at room temperature. Mix the two solutions together to obtain the transfection mixture and incubate it for further 20 minutes at room temperature. In the meantime, wash the seated CHO cells once with sterile PBS.
Replace the PBS with one ml per well of phenol red free medium, supplemented with 10%fetal bovine serum and no antibiotics. Add the entire construction mixture of one ml drop wise to each well and incubate the cells overnight at 37 degree in five percent CO2. For labeling, dilute the appropriate SNAP substrate in one mL medium supplemented with 10%fetal bovine serum to obtain a final concentration of one micromolar.
Wash the transfected cells once with PBS and add one mL per well of one micromolar SNAP substrate solution. Incubate the cells for 20 minutes at 37 degree in five percent CO2. Wash the cells thrice with phenol red free medium, and add two mL per well phenol red free medium.
Incubate the cells for 30 minutes at 37 degrees centigrade in five percent CO2. Transfer the cover slip of all samples subsequently into the imaging chamber and wash with 500 Microliter imaging Buffer. Add 500 microliter imaging buffer before moving to the FRED FCS setup.
The FRED FCS setup is equipped with a confocal microscopy water objective, two laser lines, a time quilter single foreign counting system, two hybrid BMTs, and two equities for photon collection and the data collection software. It is very crucial to align the setup every time before measurement in live cells. For adjusting focus, pinhole and coloring position, place two nanomolar green calibration solution on a glass cover slip and switch on the 485 nanometer and 560 nanometer laser operated in piles, interleaved excitation or PI mode.
Focus on the solution and adjust the pinhole and collar ring position such that the highest count rate and smallest confocal volume are obtained to get the maximum molecular brightness. Repeat this process for the red channels with 10 nanomolar red calibration solution and a mixture of both. Place 10 nanomolar DNA solution on glass cover slip, and adjust the focus of pinhole and the collar ring position such that the cross colors in between the green and red detection channels is highest.
That is source the highest amplitude. For measurements in live cells, find a suitable cell by limiting with the marker lamp and observing through the ocular. Switch on both the lasers in PI mode and focus on the membrane by looking for the maximum counts per second.
Please note, the laser power might need to be reduced for the cell samples. Preferably less than five microwatt at objective. This depends highly upon the used floral and the setup.
Observe the auto and cross coll curves of the beta-2 AR bound to EGP and snap tag probes in the online preview of the data collection software and collect several sort measurements with an acquisition time between 60 to 180 seconds. Export the correlation course and contrast from all measurements. Please take care here to correctly define the prompt and delay time windows and use some micro time getting option in the data correlation software.
In total, three different correlations are required. Auto correlation of the green channel prompt time window. Auto correlation of the red channels and the delay time window.
And finally the cross correlation of the green channel signal, and the prompt time window was the red channel signal in the delay time window. Here's the auto correlation functions of the green and red FRED for solutions and fit them to a 3 DT diffusion model with an additional triplet term of required to calibrate the shape and size of the confocal detection volume for the two use color channels, where B is the baseline of the curve and the number of molecules and focus, TD the diffusion time and S, the zero over omega zero the shape factor of the confocal volume element. The triplet blinking and photo physics is described as amplitude AR and relaxation time TR.Use the known diffusion coefficient for the green and red calibration stand outs and obtain shape factors to determine the dimensions and volume of the infective confocal volume element.
Calculate the spectrum across like IFR, the green fluorescent signal collected on channels zeroes, and two into the right detection channel, channel one and three, as a ratio of the background collected signals. Determine the direct expectation of the acceptor flow of data by the donor excitation wavelengths by the ratio of the background collected contrast of the red calibration measurements and the prompt time window excitation by green laser to the background correct account right in the delay time window excitation by red laser. Calculate the molecular brightness of B both the green and red flow force based on the background collected contrast, and to obtain number of molecules and focus on the 3 DT diffusion fit.
Fit both ultra correlations from the green and red channel, as well as across correlation from the green prompt and red delay of the double level DNA to the 3 DT diffusion model keeps the obtain shaped factors constant for the auto correlation functions. The shape factor for the cross correlation functions is usually in between those two values. Determine the amplitude at zero correlation time based on the font values of the apparent number of molecules and focus.
Calculate the amplitude ratio for a sample was a 100%co-diffusion of green and red floor force. Fit the cell samples to an appropriate model. How they're shown membrane receptor diffusion not curious in a bi-modal fashion was a short, no long diffusion time.
Additionally, the photo physics and blinking out the floor force have to be considered. Here to TD1 and TD2, are the two required to diffusion times. And a one is a fraction of the first diffusion time In contrast to the calibration measurement in which the free dice and DNA strands diffusion, all directions, membrane receptors show only 2D diffusion along the cell membranes calculate the concentration of green or red label proteins from the respect of number of molecules and focus.
And the volume of the confocal volume element, using biasing mass. Fit the two alto correlations of the double labeled sample using the same model as for the single level construct and the cross correlation function using a bi-modal diffusion model, Note for a global description of the system. All three crops have to be fit jointly.
The diffusion term is identical for all three crops. And the only difference is a reputation time falls, the cross correlation function. Calculate the concentration of green or red labor proteins from the respective number of molecules and focus and the volume of the confocal volume element.
Estimate the fraction or concentration of interacting green and red label proteins from the cell samples using the correction factors obtained from the DNA samples, the amplitude ratios of the cell sample and the respective obtained concentrations. Fit the two alter correlations of the FRED sample as a single labeled samples and the front cross correlation to a bi-modal diffusion model containing an anti correlation term, where AF reflects the amplitude of the total anti correlation and AR and TR the respective amplitude and relaxation time. In case of anti-correlated fluorescence changes due to FRED one or several anti correlation terms might be required, which resulted in a dip of the Fred cross correlation function at low correlation times coinciding with a rise in the two auto correlation functions.
The calibration measurements of the green and red floral solutions to shave factors of 6.5 and the green channels, and 6.8 in the red channels. Thus, the confocal volume has the size of 1.4 and 1.9 femto later on this measurement day, the molecule brightness lies at 12.5 kilohertz per molecule, and 2.6 kilohertz per molecule. We have 15%of crosstalk from the green floral for into the red channels after donor excitation and 38%of direct except excitation of the red floral form by the green, from our DNA measurement, we determine the correction factors for the confocal overlap volume to 0.6 and 0.7 too, the cells transfected with single label construct show bi-modal two dimensional diffusion on the cell membrane where roughly half of the molecules diffuse slowly on the 50 to a hundred millisecond timescale and the other half relatively fast around two milliseconds in both constructs.
Additionally, triplet blinking is present. However, in the red level snap construct another relaxation time is acquired at 180 microseconds, which might stem from Unbound snap straight. From the double label itself, transected with the anti snap construct where the two labels on two different sides of the cell membrane, two less measurements have been collected the noise and the red coat onto correlation.
And the PI cross correlation is quite high here. Here 70%of the molecules, if you slowly under a hundred millisecond timescale, and only 30%fast around one millisecond, the fraction of double liberate molecules is low and lies between 15 to 25%The data for the CT snap looks better and it's less noisy. However, the expected front deepest anti correlation cannot be seen and is most likely mass by the high amount of crosstalk, direct accept excitation and triplet blinking of both floral force, and once data analysis schemes taking advantage of the collected fluorescence intensity DKS might help to rescue this as shown for the simulated data set.
The advantage of Fred FCS technique is that along with mobility, we can investigate the conference dynamics of GPCRs. However, performing FRED FCs in labs is challenging and request good transfected cells, efficient labeling and a perfect calibrated setup. Just a FRED pair pro force is also very crucial.
Critical experimental steps include the optimization of transfection optimization, the minimization of background, and the auto fluorescence. Additionally and Atoms analysis pipeline will help to understand the various dynamics of preceptors in live cells. We hope that this protocol will be useful perform the FRED FCS approach in live cells experiments.
