The overall goal of this experiment is to visualize and quantify compartmentalized translation events taking place during the initial steps of cell adhesion. This method has broad application. Use it whenever rapid specific translation response might be assay such as when analyzing cell migration, adhesion, or early drug response.
The main advantage of this technique are that, it is easy, rapid, and cost-effective. It only requires puromycin, antibodies directed against puromycin, and the standard confocal scan imaging system. To begin, make a suspension of MRC-5 cells.
Use Trypsinization, followed by centrifugation, removal of the supernatant, then resuspension in DMEM with ten percent FBS, at 40 thousand cells per milliliter. Incubate the suspension with gentle rotation for 20 minutes to completely dissociate the focal adhesion complexes. This is critical.
Next, into two 35 millimeter glass-bottom dishes, add two milliliter aliquots suspension, and incubate them to allow for cellular adhesion and SiC formation. After 40 minutes, treat one plate with cycloheximide, and return it to the incubator. This plate will function as the negative control.
After 55 minutes, apply puromycin to both plates at the predetermined concentration, and incubate the plates for five more minutes. After one hour of seeding and five minutes of puromycin in cooperation, wash each plate twice with two milliliters of ice-cold PBS. Then fix the cells with one milliliter of four percent formaldehyde in PBS for 15 minutes at room temperature.
After the fixation, wash the cells three times with PBS, then permeabelize the cells using 500 microliters of 0.5 percent triton X-100 in PBS for 20 minutes at room temperature. Next, wash the plates three times with PBS, and then apply 500 microliters of one percent BSA in PBS. Continue the room temperature incubation for 20 more minutes.
To remove excess BSA, wash the cells three times with 0.1 percent Tween-20 in PBS. Now, incubate the cells with 300 microliters of anti-puromycin antibody. Remove the unbound antibody using three washes with 0.1 percent Tween-20.
Next, apply three hundred microliters of a mixture of two fluorophore-conjugated secondary antibodies to visualize puromycolated polypeptides and de factem. Incubate the cells for an hour at room temperature in the dark. After an hour, wash the plates three times to remove the unbound antibodies and then apply three hundred microliters of 0.1 percent Tween-20 solution, supplemented with DAPI.
Allow the DAPI to stay in the cells for five minutes at room temperature. Finally, wash the cells four more times, finishing with a wash in pure PBS. Then, store the cells in two milliliters of PBS for imaging.
Using any commercially available confocal imaging system, first determine the appropriate settings that maximize the dynamic range for quantification. Focus on quantification can only be obtained if peak size saturation is avoid and the concentration is adjusted properly. The setting can be attained by carefully adjusting the laser power advantage and offsets.
First, adjust the zoom factor and the number of pixels to optimize the pixel size. Do so according to the Nyquist theorem. Next, decrease the scan speed and use averaging to improve the signal to noise ratio.
Then, manually determine the appropriate top and bottom focal planes along the Z axis, and set the StepSize by calculating the axial resolution and dividing this distance by 2.3. The typical result is 20 to 25 image layers. Once the settings are tweaked, acquire a confocal image of an entire single cell using a 60x Plan Apo oil-immersion objective with a 1.42 numerical aperture.
To quantify and analyze the flurophore signals, first open an image corresponding to the z plane layer of interest in image J.Then, using the drawing tool, make a line through the cell where quantification is desired. Next, modify the line whiff by double-clicking on straight. The intensity values will be averaged through the width of the line, so use a thinner line to avoid signal overlap from different structures.
Then, profile the signal density along the line. Now, repeat the process of collecting the signal density along the same line in the channel that corresponds to a flurophore. The values will always be ordered using the orientation of the line.
Now, copy the profile data and paste it in a spreadsheet for analysis. Repeat this process of collecting data from every Z plane image of interest before proceeding with the statistical analysis. Alternatively, instead of collecting signal along an axis, signal can be collected from an area of the image.
First use the geometrical form function to select a region of interest. Next, adjust the threshold level to avoid any background. In the histogram of pixel intensities, move the slider to adjust which pixels will be included in the quantification.
Set the threshold to eliminate all the red pixels outside the cell. Now, define the measurement parameters needed to measure the signal of interest and not background signal. Toggle the limit to threshold and integrated density options.
Next, use the measure command. This will open a new window with the mean gray values and pixel count in the selected area. Now repeat the process, applying the same threshold to determine the signal intensity in the entire cell.
Then use this data to calculate the ratio of signal in the selected area to that of the whole cell. For accurate measurement of translation events using purmycin-incorporation, the optimal conditions for an axial measurement were first assessed. A spectrum of concentrations versus a five or ten minute treatment was compared.
The acceptable puromycin concentration for MRC-5 and HeLa cells was a little higher than what is needed for Huh-7 cells. Cells treated for ten minutes with high puromycin concentrations generated mostly smaller puromycolated polypeptides. The smaller polypeptides defused more rapidly, and are not desirable.
The shorter incubation time was thus deemed more desirable. Cycloheximide treatment is an effective and important negative control. In MRC-5 cells, only a weak puromycin signal was detected after treatment with cycloheximide.
Using axial signal quantification, the general localization of puromycolated polypeptides corresponding to newly-synthesized proteins could be assessed at different levels within the cell. A quantitative distribution of the signal throughout the cell was also feasible. For either technique, it was important to quantify the signal for every Z plane in the image stack to accurate assess translation events in a whole cell.
After watching this video, you should have a good understanding on how to evaluate translation event in sub-cellular compartment. Once mastered, this technique can be done within a day, if performed properly. When one thinks this procedure, it's important to remember that the concentration of puromycin and its incubation time is important.
To limit the diffusion, it is preferable to limit the incubation time. It is also important to recognize that the quality of the image acquisition type is a critical to obtain good results. Don't forget that working with formaldehyde can be extremely dangerous and precautions such as working in a chemical hood should always be taken while performing this procedure.
