Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

This protocol allows for the real-time assessment of various processes and biochemical parameters within the lumen of individual macropinosomes. These methods can be employed to drug discovery in the cell biology of macropinocytosis. This protocol provides the advantage of revealing heterogeneity at both the cellular and organelle level.

One day before the assay seed Raw264.7 cells at a density of five times 10 to the power of the cells in 100 microliters per well, in a glass bottom 96-well plate with black sides. On the day of the assay check whether the wells are at least 70%confluent. Then wash all the wells with 100 microliters of HBSS, prewarmed at 37 degrees Celsius.

Next, add 100 microliters of HBSS containing 0.025 milligrams per milliliter of TMR labeled 70 kilodalton dextran and 0.025 milligrams per milliliter of fluorescein labeled 70 kilodalton dextran in each well, then incubate the cells at 37 degrees Celsius for 15 minutes. After the incubation is complete, remove the plate from the incubator and wash the cells six times with 100 microliters of HBSS. After the last wash add 100 microliters of prewarmed HBSS to the cells then place the plate on a microscope with a heated stage and chamber.

After adjusting the excitation and emission parameters for TMR and fluorescein as needed, acquire an image from each well alternating between the two fluorophores in between each well. After the first acquisition, check whether all the wells remained in focus across the entire plate, then acquire images of each well at one to 15 minute intervals for the desired length of time. During the image acquisition, adjust the pH of a 50 milliliter aliquot of a potassium rich solution buffered with 25 millimolar HEPES to 7.5 using 10 molar hydrochloric acid, or 10 molar potassium hydroxide as needed.

Then adjust the pH of 350 milliliter aliquots of the potassium rich solution buffered with 25 millimolar MES to pH 6.5, pH 5.5 and pH 5.0. Once the image acquisition is complete, remove the HBSS from the 96-well plate containing the cells and add the potassium rich solution at pH 7.5. Then add Nigericin at a final concentration of 10 micrograms per milliliter.

Place the plate back on the microscope and acquire images of each well using the same acquisition settings as demonstrated previously. To measure oxidative events within macropinosomes, seed RAW264.7 cells in a 96-well plate one day before the assay as demonstrated previously. Then on the day of the assay wash all the wells with 100 microliters of HBSS prewarmed to 37 degrees Celsius.

Next, add 100 microliters of HBSS containing 0.025 milligrams per milliliter of TMR labeled 70 kilodalton dextran and 0.025 milligrams per milliliter of H2DCFDA labeled 70 kilodalton dextran to each well, then incubate the cells at 37 degrees Celsius for 15 minutes. After removing the plate from the incubator wash the cells six times with 100 microliters of HBSS. After the last wash, add 100 microliters of HBSS to each well and place the plate on the microscope.

After adjusting the excitation and emission parameters for TMR and H2DCFDA as needed, acquire images of each well at one to 15 minute intervals as demonstrated previously. To measure protein digestion within macropinosomes, seed RAW264.7 cells one day before the assay, as demonstrated previously. Then on the day of the assay wash all the wells with 100 microliters of HBSS prewarmed at 37 degrees Celsius.

Next add 100 microliters of HBSS containing 0.025 milligrams per milliliter of TMR labeled 70 kilodalton dextran in 0.2 milligrams per milliliter BODIPY labeled ovalbumin to each well. Then incubate the plate at 37 degrees Celsius for 15 minutes. After removing the cells from the incubator, wash them six times with 100 microliters of HBSS.

After the last wash add 100 microliters of HBSS to each well, then place the plate on the microscope. After adjusting the excitation and emission parameters for TMR and BODIPY as needed, acquire images of each well at one to 15 minute intervals as demonstrated previously. When measuring the pH, oxidative events or protein degradation in macropinosomes, the dynamics of acidification cannot be measured for a certain time period corresponding to the dextran loading phase, which varies depending on the cell type used.

When measuring the macropinosome pH, the fluorescein to TMR ratio will become progressively smaller and will plateau within the 15 minutes of acquisition. This plateau corresponds to a pH of approximately five, which roughly matches the pH of lysosomes. At the end of each acquisition, an In situ calibration is performed.

The fluorescein to TMR ratio should be largest within the calibration buffer at pH 7.5 and become progressively smaller as the calibration buffers become more acidic. This allows for the generation of a calibration curve. When measuring oxidative events within macropinosomes, the ratio of H2DCFDA to TMR will become progressively larger and will likely plateau within the first 20 to 30 minutes in an activated RAW264.7 cells.

When measuring macropinosomal protein digestion, a progressively strong fluorescein signal will be liberated as the ovalbumin is digested. In RAW264.7 cells, the increase in fluorescence liberated from the digested BODIPY labeled ovalbumin, will plateau within the first 30 minutes. With this emerging role in homeostasis and it's newly discovered relevance to cancer pathology, a renaissance macropinocytosis research is currently underway.

These methods provided to a tool box for exploring the contribution of macropinocytosis to these fields of study.