Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is an endocytic pathway that is important for a variety of cellular processes, including cancer metabolism. This protocol allows you to quantify the extent of macropinocytosis in cells in vitro. The automation is aimed at maximizing reproducibility, productivity and reducing experimental variation.

Moreover, the fluorescent microscopy allows you to visually inspect the sample to determine experimental quality and assess additional macropinosome characteristics. Demonstrating the procedure with me will be Cheska Marie Galapate, a research assistant from my laboratory. For the 24-well plate with coverslip format, use forceps to grip a single coverslip from the ethanol bath.

Tap the coverslip to the inside wall of the plate to remove excess ethanol and place the coverslip flat on the bottom of the well. After the ethanol evaporates, wash the coverslip twice with DPBS, then seed the cells on top of the coverslip by adding 500 microliters of the cell suspension to each well. Place the cells in a 37 degree Celsius cell incubator with 5%carbon dioxide until cell confluency reaches 60 to 80%on the day before macropinosome labeling.

The day before macropinosome labeling, replace the media in the wells with 500 microliters of pre-warmed serum-free media and place the cells in the incubator for 16 to 24 hours. For the 96-well microplate format, begin by transferring the cell suspension to a 25 milliliter reagent reservoir. Then using a multichannel pipette, seed 100 microliters of the cell suspension to each well of a black 96-well high-content screening microplate with an optically clear cyclic olefin or glass bottom.

Incubate the cells until cell confluency reaches 60 to 80%on the day before macropinosome labeling. The day before macropinosome labeling, remove and discard the media from each well using a multichannel aspiration adaptor for standard tips attached to a vacuum pump. Alternatively, a multichannel pipette can be used.

Using a reagent reservoir and multichannel pipette, gently add 100 microliters of pre-warmed serum-free media to each well, then place the cells in the cell incubator for 16 to 24 hours. For the 24-well plate with coverslip format, replace the media in the wells with 200 microliters of serum-free media with fluorophore-labeled high molecular weight dextran and place the cells in the cell incubator for 30 minutes. After the incubation, aspirate the media and gently but quickly wash the cells five times with ice cold PBS using a pre-cooled wash bottle.

Firmly shake the plate by hand during washes to aid in dislodging dextran aggregates that become stuck to the coverslips. Next, fix the cells by adding 350 microliters of 3.7%formaldehyde and incubating for 20 minutes, then aspirate the fixation solution and wash the cells with PBS twice. Stain the nuclei with 350 microliters of DAPI in PBS.

After 20 minutes, aspirate the DAPI solution and wash the cells with PBS thrice. Adhere silicone isolators side by side on a microscope slide to obtain even spacing and reproducible localization of the coverslips required for imaging automation. Then for each coverslip, add a drop of hardening fluorescence mounting media on the microscope slide within the open space of the isolator.

Pick up a coverslip using forceps and remove excess PBS by gently tapping the side of the coverslips on a lint-free wipe. Next, place the coverslip upside down on the drop of mounting media and gently tap the coverslip using closed forceps to remove bubbles from the mounting media. Store the slides in a dark environment and allow the mounting media to dry at room temperature, typically taking 16 to 24 hours.

Before imaging, remove the isolators from the microscope slide. After the slides have been equilibrated to room temperature, clean the coverslips using a cotton tipped applicator wetted with ammonia-free glass cleaner. Subsequently, use a clean cotton tipped applicator wetted with 70%ethanol to clean the coverslip and leave it dry.

For the 96-well microplate format, after aspirating the wells as demonstrated previously, add 40 microliters of serum-free media with fluorophore-labeled high molecular weight dextran to the wells, then incubate the cells in the cell incubator for 30 minutes. After incubation, discard the media in the microplate by manually flicking the plate upside down into an empty five liter beaker, then rinse the cells and the microplate twice by slowly submerging the plate vertically at a slight angle into a two liter beaker filled with ice cold PBS and subsequently discarding the PBS in the microplate by flicking the plate upside down into the five liter beaker. After the last PBS rinse, fix the cells by adding 100 microliters of 3.7%formaldehyde in PBS to each well using a 25 milliliter reagent reservoir and a multichannel pipette.

After a 20-minute incubation at room temperature, remove the fixation solution and wash the cells with PBS using the submerging and flicking technique. After the second PBS wash, stain the nuclei with 100 microliters of DAPI in PBS per well. After 20 minutes, rinse the cells thrice with ice cold PBS as demonstrated previously.

Remove any residual PBS by tapping the microplate upside down onto a lint-free wipe. Then add 100 microliters of fresh PBS to each well using a 25 milliliter reagent reservoir and multichannel pipette. Before imaging, let the plate equilibrate to room temperature, then wipe the cell culture plate dry with a lint-free wipe.

Alternatively, store the plate covered away from light at four degrees Celsius for up to one week. For automated macropinosome imaging, create an automation protocol to acquire the images with a 40X air objective in the wavelength channel of the dextran fluorophore and DAPI. Next, optimize exposure settings using a sample predicted to have the highest level of macropinocytosis to avoid over-exposure, which may result in saturation of the signal and loss of intensity data.

Use focus settings that readily and consistently locate the sample to produce high-quality images. Acquire multiple images across each well or coverslip to account for sample variability and obtain an accurate representation of the sample. To determine the macropinocytic index, subtract the background for the DAPI and corresponding dextran image by applying the appropriate function, frequently called the rolling ball function.

Adjust the settings so that the background noise is minimized with minimal to no subtraction effect on the DAPI and dextran signal. Next, using a field with high dextran signal, determine the intensity signal settings, frequently called the threshold function, to select the nuclei, then determine the minimum intensity signal setting required to select only the macropinosomes. For the dextran image, calculate the total fluorescence within the created macropinosome selection or use the selection to determine the total area positive for dextran.

For the DAPI image, use the selection to determine the number of nuclei in the image to reflect the number of cells present. Then determine the macropinocytic index by dividing the total dextran fluorescence or area by the number of cells determined by DAPI. In AsPC-1 PDAK cells, adding EGF at 100 nanograms per milliliter for five minutes before adding the dextran activates macropinocytosis.

Moreover, autocrine EGF activation of macropinocytosis can be induced by glutamine deprivation for 16 to 24 hours. MIA PaCa-2 cells show constitutive macropinocytosis which is inhibited by a 30-minute treatment with 75 micromolar EIPA or a two-hour treatment with 10 micromolar EHop-016. In a dose response experiment, the macropinocytic index gradually decreased at higher drug concentrations of EHop-016 and EIPA, thereby confirming the existence of constitutive Rac1-dependent macropinocytosis.

You can adapt this procedure to assess macropinocytosis of other fluorescently tagged cargo such as albumin. In addition, it is recommended to evaluate whether macropinocytic uptake of albumin contributes to cell fitness by performing cell viability assays. This technique has been central to the identification of macropinocytic nutrient supply in cancer and stromal cells and the automation has greatly increased our condition testing capacity.