Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The overall goal of the following experiment is to quantitatively assess the spatial colocalization of target pairs, in this case, examining drug-induced receptor trafficking. This is achieved by treating live cultured cells with drug to induce altered receptor trafficking. As a second step, double immuno labeling of the target receptors and intracellular trafficking compartments allow spatial localization of the target pairs by multichannel confocal microscopy be next.

Quantitative colocalization analysis is used to measure the tendency of the target receptors and compartments to be found in the same place. The results show changes in receptor trafficking following drug treatment based on the colocalization of receptors and intracellular trafficking compartments. The main advantage of this technique over existing methods like overlay colocalization, is that it provides quantified measures of colocalization, which then allows far more powerful analyses and complex experimental designs.

Though this method can provide insight into receptor trafficking, it can also be applied to changes in the spatial colocalization of just about any two proteins in cultured cells. To begin this procedure, remove the original growth medium and replace it with medium containing the drug of interest at the chosen concentration. Next, incubate the cells in the original growth conditions for the chosen treatment duration.

After incubation, wash the cells gently with one milliliter of washing buffer each time for three times. Fix the cells with 300 microliters of 4%para formaldehyde in 0.1 molar PBS for 10 minutes at 37 degrees Celsius. Then wash the cells again with one milliliter of washing buffer each time for three times.

Subsequently, incubate the cells in 300 microliters of blocking buffer for two hours at room temperature. Afterwards, incubate the cells with primary antibody in 300 microliters of antibody diluent for 48 hours at four degrees Celsius. Then wash the cells gently with one milliliter of washing buffer each time for three times.

Next, incubate the cells with secondary antibody conjugated to Fluor in 300 microliters of antibody diluent for one hour of room temperature and protect the cells from light. After one hour, wash the cells gently with one milliliter of washing buffer each time for three times. To remove the cover slip from the 24 well plate hold the plate of 45 degrees.

Place the tip of a pair of fine forceps at the top edge of the cover slip where it meets the well floor. Then gently tilt the cover slip in the washing buffer away from the well floor. The cover slip will come to rest against the well wall where it can easily be removed with forceps.

Then mount the cover slips with the cells facing down on microscope slides with anti fading mounting medium using a 100 x high magnification objective. First, locate a representative cell to be imaged using epi fluorescence. Next, configure the image capture settings Record eight bit uncompressed tiff images of each labeled channel to image each channel sequentially and to average four to six scans for the final image.

Then switch to the confocal imaging path and focus on a Z plane through the center of the cell. Optimize the pinhole laser power and photo multiplier tube voltage and offset for each channel. Record these settings and use the same settings for imaging all the cells labeled for any given target pair.

In the same Replicate next locator cells to be imaged with a high magnification objective. Using epi fluorescence, switch to the confocal imaging path and focus on a Z plane through the center of the cell if available. Use software zoom crop function to restrict the scan area to the cell of interest.Afterwards.

Capture images for each labeled channel using the settings previously established, and repeat the procedures to image 15 or more cells per condition per replicate. To ensure sufficient sample collection. In this procedure, open the pair of images of a cell in image J.Use the image color merge channels command to generate an RGB image.

Then draw around the cell of interest. Use the image j plugin the SC colocalization to quantify colocalization of the targets in the selected cell. Record the desired colocalization measure and repeat the procedure for each image cell.

Next, calculate the mean of the recorded colocalization values of the common control conditions of each replicate of each labeling condition. Multiply the means by minus one to yield the offset for each replicate in each labeling condition. Then add the offset for each replicate in each labeling condition to each colocalization value in that replicate.

This will normalize the data such that the mean colocalization measure for the common control condition is zero in each replicate of each labeling condition. After that, all the data from all replicates of each labeling condition will allow the analysis of changes in colocalization across replicates Shown here are the primary cultures of dorsal root ganglia sensory neurons that were exposed to prolonged and acute drug treatments. The cells were then immuno labeled for delta opioid receptors and a marker of recycling endosomes with distinct primary secondary antibody pairs and were imaged by two channel sequential confocal microscopy.

In addition to subsequent quantitative colocalization and analysis, these representative images were processed to generate false color colocalization maps shown here. The mean colocalization scores for delta opioid receptors and markers of early endosomes recycling endosomes and lysosomes receptor colocalization with each compartment are compared between groups receiving different acute drug treatments and drug-induced changes in receptor trafficking are observed. While attempting this procedure, it’s important to remember to acquire high quality consistent micrographs to allow valid quantitative analysis.

After watching this video, you should have a good understanding of how to perform quantitative colocalization analysis.