Glutamine Flux Imaging Using Genetically Encoded Sensors

The overall goal of this procedure is to image glutamine transport via a heterologous expressed transporter in live cells using firster resonance energy transfer or fret sensors. This is accomplished by first transecting cause seven cells with a glutamine sensor and a transporter. The sensor consists of a fret donor inserted into a bacterial glutamine binding protein and a fret accepter at the C terminus of the glutamine binding protein.

The second step is to set up a perfusion system and image the cells in real time using an inverted fluorescent microscope. The final step is to analyze the images and visualize glutamine dynamics. Ultimately, transporter activities in a single cell can be detected using these fret sensors.

The main advantage of this technique over existing methods like chromatography based measurement, is that you get much higher spatial temporal resolution. This technique consists of three parts, transfection of cells with sensor proteins, real-time imaging, and post experimental analysis Cost. Seven cells are used in this experiment due to their low endogenous glutamine transport activity.

Seed the cells at about 70 to 80%co fluency in DMEM plus 10%cosmic calf serum or CCS and 100 units per milliliter. Penicillin streptomycin in an eight well glass bottom dish. Incubate at 37 degrees Celsius, 5%CO2, 100%humidity for 24 hours.

After 24 hours. Replace the growth medium with DMEM plus 10%CCS without antibiotics grow overnight at 37 degrees Celsius, 5%CO2 100%humidity on the day of transfection. Add 0.4 micrograms each of the plasma DNA encoding the glutamine sensor and the tagged obligatory amino acid exchanger m cherry A SCT two to 50 microliters of serum free media.

Add one microliter of transfection reagent to 50 microliters of serum free media mix gently and incubate at room temperature for five minutes. Next, mix the solution containing the DNA with the diluted transfection reagent and incubate at room temperature for 20 minutes. After 20 minutes, gently add the transfection reagent mixture on top of the cost seven cells and mix gently by rocking the chamber.

Incubate for 24 hours at 37 degrees Celsius, 5%CO2, and 100%humidity after 24 hours. Replace the medium with DMEM plus 10%CCS plus 100 units of penicillin streptomycin. Return the cells to the incubator prior to starting the perfusion.

Prepare perfusion buffers A through F following the recipes in the accompanying manuscript. Attach stop Cox to 50 milliliter syringes. Close the stop cocks and fill the syringes with the perfusion solutions.

Open the stop cock for a brief period to fill the headspace in the stop cock with the buffer. Next switch on the eight channel gravity. Feed perfusion system with a perfusion pencil and select manual mode.

Under the select function option. Place a waste container under the perfusion pencil. For the success of this experiment, it is important to set up the perfusion in a way that minimizes the drift and the solution carry over.

Manually activate the ports by pressing the corresponding numbers and fill the tubings with water. Using a 10 milliliter syringe, close the ports, set the syringes containing buffers A through F on ports one through six of the perfusion system, and open the stop cocks. After that.

Open the ports again to replace the water in the tubing with the buffers. The flow rate should be around 0.8 milliliters per minute. Press cancel once on the perfusion controller to go back to the select function menu.

Toggle to the edit program option, then type in the perfusion protocol indicated in this table. Save the perfusion program. Cells are imaged 48 to 72 hours Post transfection.

Wash the wells of the glass bottom dish twice with perfusion buffer A taking care to not wash off the cells. Set the glass bottom dish on the stage of the fluorescent microscope. Connect the perfusion system to the chamber.

If an open chamber is used, set up another pump that removes the perfusion solution from the chamber. Open the slide book imaging software. Start a new slide using the focus function, find cells coex expressing the sensor and A SCT two M cherry using appropriate filter sets.

Adjust the gain by clicking the camera tab and sliding the slide bar under gain. Also, adjust the neutral density filter setting from the neutral density filter dropdown menu. Click image capture, and then in the acquisition window, specify the filters to be used.

Images will be acquired in three channels. Donor excitation, donor emission donor excitation, acceptory mission and acceptor excitation acceptory mission. Click the test button to adjust the exposure time by typing the desired exposure time in the box Next to the filter settings, all three channels need to be exposed for the same length of time.

Draw a region of interest using the regions tool. Select time-lapse in the capture type box. Then specify the interval and time points for the experiment.

Next, draw a region in an area without any fluorescent cells to be used for background subtraction. Right click the region drawn and then set it as a background. Ideally, cells that are not expressing the sensors should be used as the background region to account for both the autofluorescence and background fluorescence detected by the camera.

Select the desired program under select function run programs option, toggle to the saved program and then hit enter on the perfusion controller. The program is now loaded and hitting any key will start the perfusion to ensure that both the perfusion and the imaging experiment start at the same time. Click the start button in the capture window at the same time as pressing a key on the perfusion controller.

Shown here are typical time course experiments using cells coex expressing the a s CT two transporter and fret glutamine sensors with affinities of eight millimolar and 100 micromolar. Influx of glutamine is detected as the change in fluorescence intensity ratios between the donor mtfp one and the acceptor Venus. Influx of glutamine in the presence of alanine is also demonstrated substrate.

Specificities of transporters can also be examined using the sensors. Here, cells were preloaded with glutamine and then various amino acids were added to the extracellular perfuse. Eight as expected A ct two substrates induced glutamine flx, whereas nons substrate amino acids did not.

The absence of fret efficiency changes upon addition of a substrate can be due to several reasons, such as the low uptake capacity for the substrate being tested as shown in cells expressing the eight millimolar sensor or the 100 micromolar sensor. But that do not express the a s CT two transporter to confirm that the fret efficiency change is due to the actual change in the substrate and not the change in other parameters. The same perfusion protocol is performed using cells expressing a sensor that is expected to be saturated under the experimental condition.

And this example, the affinity of the sensor is 1.5 micromolar. After watching this video, you should have a good understanding of how to image the dynamics of metabolites using fret sensors. Don't forget that cell culture should be treated appropriately following the standard operating procedures so that the risk of exposure is minimized.