Name: measuring the ph, redox chemistries, and degradative capacity of macropinosomes using dual-fluorophore ratiometric microscopy
Uploaded: 2021-08-19T13:30:00
Description: Watch this Scientific Journal Video about Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy at JoVE.com

We thank the University of Calgary for its support. We would also like to thank Dr. Robin Yates for access to reagents, equipment, and useful discussions.

1. Preparation of cells
<ol>
	<li>Grow Raw264.7 cells at 37 &#176;C and 5% CO2 to 70% confluency in RPMI supplemented with 10% heat-inactivated serum.</li>
	<li>One day before the assay, seed Raw264.7 cells at a density of 5 x 104 cells per well in a 96-well plate. Ensure that each well contains 100 &#956;L of growth medium. Ensure that the 96-well plate has black sides and a glass-bottom for imaging. 
	NOTE: Here, 96-well plates were used for imaging. The 96-well plates allow for the use of ...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLIT2/ROBO1-signaling+inhibits+macropinocytosis+by+opposing+cortical+cytoskeletal+remodeling.">SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling.</a> Nature Communications. 11, 4112 (2020).</li><li>Liu, Z., Roche, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+in+phagocytes:+regulation+of+MHC+class-II-restricted+antigen+presentation+in+dendritic+cells.">Macropinocytosis in phagocytes: regulation of MHC class-II-restricted antigen presentation in dendritic cells.</a> Frontiers in Physiology. 6, 1 (2015).</li><li>Desai, A. S., Hunter, M. R., Kapustin, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+macropinocytosis+for+intracellular+delivery+of+therapeutic+nucleic+acids+to+tumour+cells.">Using macropinocytosis for intracellular delivery of therapeutic nucleic acids to tumour cells.</a> Philosophical Transactions of the Royal Society of London B. Biological Sciences. 374, 20180156 (2019).</li><li>Freeman, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-gated+monovalent+ion+fluxes+regulate+endocytic+traffic+and+support+immune+surveillance.">Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance.</a> Science. 367, 301-305 (2020).</li><li>Canton, J., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+phagosomal+pH+by+fluorescence+microscopy.">Measuring phagosomal pH by fluorescence microscopy.</a> Methods in Molecular Biology. 1519, 185-199 (2017).</li><li>Cheung, S., Greene, C., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+multiple+lumenal+parameters+of+individual+phagosomes+using+high-content+imaging.">Simultaneous analysis of multiple lumenal parameters of individual phagosomes using high-content imaging.</a> Methods Molecular Biology. 1519, 227-239 (2017).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+size+of+fluorescent+dextran+on+its+endocytic+pathway.">The effect of the size of fluorescent dextran on its endocytic pathway.</a> Cell Biology International. 39, 531-539 (2015).</li><li>Canton, J., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+lysosomal+pH+by+fluorescence+microscopy.">Measuring lysosomal pH by fluorescence microscopy.</a> Methods in Cell Biology. 126, 85-99 (2015).</li><li>Armstrong, J. K., Wenby, R. B., Meiselman, H. J., Fisher, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamic+radii+of+macromolecules+and+their+effect+on+red+blood+cell+aggregation.">The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation.</a> Biophysical Journal. 87, 4259-4270 (2004).</li><li>Blum, J. S., Wearsch, P. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+controls+phagosomal+pH+and+antigen+cross-presentation+in+human+dendritic+cells.">NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.</a> Blood. 112, 4712-4722 (2008).</li><li>Canton, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+receptor+DNGR-1+signals+for+phagosomal+rupture+to+promote+cross-presentation+of+dead-cell-associated+antigens.">The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens.</a> Nature Immunology. 22, 140-153 (2021).</li><li>Mishra, R., Bhowmick, N. 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Although there are a number of protocols for both low and high-throughput measurements of macropinocytic uptake in macrophages, fibroblasts, and even Dictyostelium spp.3,7,31,32,33, very few attempts have been made to measure the luminal biochemistry of these dynamic compartments. This is likely due to a paucity of probes that can be efficiently targe...

Macropinocytosis refers to the uptake of large quantities of extracellular fluid into membrane-bound cytoplasmic organelles called macropinosomes1,2. It is a highly conserved process performed by free-living unicellular organisms, such as the amoeba Dictyostelium spp.3, as well as anthozoans4 and metazoans2. In most cells, macropinocytosis is an induced event. The ligation of cell-surface receptors induces the protrusion of actin-driven plasma membrane extensions referred to as ruffles. A fraction of those ruffles, by some poorly understood mechanism, seal at their distal tips to form macropinosomes (although beyond the scope of this methods paper, for detailed reviews on the mechanics of macropinocytosis, please refer to references1,2,5,6,7). The extracellular stimulus inducing macropinocytosis is most often a soluble growth factor5,8. Accordingly, the macropinocytic event allows for the ingestion of a bolus of extracellular material from which the cell can derive useful metabolites to facilitate growth. Unfortunately, this pathway for nutrient delivery can also drive pathology. Certain cancer cells harbor mutations that result in continuous or constitutive macropinocytosis. The continuous delivery of nutrients facilitates the uncontrolled proliferation of cancer cells and has been linked to particularly aggressive tumors9,10,11,12,13. Similarly, viruses can induce macropinocytosis to gain access to host cells, thereby driving viral pathology14.
Macropinocytosis also functions in the maintenance of immunity to pathogens. Certain innate immune cells such as macrophages and dendritic cells engage in the constitutive and aggressive sampling of extracellular fluid via macropinocytosis6,15,16. This mode of macropinocytosis is incredibly active, and a single dendritic cell can engorge itself with a volume of extracellular fluid equivalent to its own weight every hour17. Despite this constitutive sampling, macrophages and dendritic cells do not replicate uncontrollably as do tumor cells, instead, they seem to process the extracellular material in such a way that information can be extracted to inform upon the presence, or indeed absence, of potential threats. Information is extracted as i) pathogen-associated molecular patterns that can be read by intracellular pathogen recognition receptors and ii) short stretches of amino acids that can be loaded onto major histocompatibility molecules for screening by cells of the adaptive immune system16,18,19. Whether pathogens subvert this pathway for information processing by immune cells is at present unclear.
Despite these well-defined and critical roles for macropinocytosis in both the maintenance of immunity and homeostasis and in contrast to other more commonly studied modes of endocytosis, little of the inner (luminal) workings of macropinosomes is known. Developing standardized protocols and tools to study the luminal biochemistry of macropinosomes will not only help us to understand their unique biology better but will provide insight that can be leveraged for novel therapeutic strategies, including drug delivery20. This method manuscript will focus on recently developed tools to dissect, at the single organelle level, various aspects of the luminal biochemistry of macropinosomes.
Fluorophores can be used to measure specific biochemistries of organelles if i) they partition preferentially into the compartment of interest and/or ii) they undergo spectral changes in response to the parameter of interest. For example, in the case of pH, fluorescent weak bases, such as acridine orange, cresyl violet, and the LysoTracker dyes accumulate preferentially in acidic organelles. Therefore, their relative intensity is a rough indication that the labeled organelle is acidic. Other pH-responsive fluorophores, such as fluorescein, pHrodo, and cypHer5e, undergo spectral changes upon binding to protons (Figure 1A-C). Changes to the fluorescence emission of pH-sensitive fluorophores can therefore provide a useful approximation of pH. The use of single fluorophores, however, presents a number of disadvantages. For example, changes to the focal plane, photobleaching, and changes to the volume of individual organelles, a common occurrence in macropinosomes21, can induce changes to the fluorescence intensity of single fluorophores, and this cannot be easily corrected for22. Single-wavelength assessments, although useful for visualizing acidic compartments, are therefore purely qualitative.
A more quantitative approach is to target the parameter-sensitive fluorophore along with a reference fluorophore to the organelle of interest. The reference fluorophore is ideally insensitive to biochemical changes within the organelle (Figure 1D-F) and can therefore be used to correct for changes in the focal plane, organellar volume, and, to some extent, photobleaching23. Using this approach, referred to as dual-fluorophore ratiometric fluorescence, correction can be achieved by generating a ratio of the fluorescence emission of the parameter-sensitive fluorophore to the reference fluorophore.
Here, the protocol will be utilizing the principle of dual-fluorophore ratiometric imaging to measure pH, oxidative events, and protein degradation within macropinosomes. In each case, a fluorophore will be selected that is sensitive to the parameter of interest and a reference fluorophore. In order to target the fluorophores specifically to macropinosomes, they will be covalently coupled to 70 kDa dextran, which is preferentially incorporated into macropinosomes24. All assays will be performed in Raw264.7 cells but can be adapted to other cell types. Where possible, the fluorescence ratios will be calibrated against a reference curve to gain absolute values. Importantly, all measurements will be performed in live cells for dynamic and quantitative assessment of the luminal environment of macropinosomes.
When selecting pH-sensitive fluorophores, a number of considerations must be weighed. The first is the pKa of the fluorophore, which indicates the range of pH values at which the probe will be most sensitive. If it is assumed that shortly after formation, the pH of the macropinosome will be close to that of the extracellular medium (~pH 7.2) and that it will progressively acidify through interactions with late endosomes and lysosomes (~pH 5.0), then a probe with a pKa that is sensitive within that range (Figure 2C) should be selected. The fluorophore fluorescein, which has a pKa of 6.4, is optimally sensitive within that range. It has been used extensively to measure other similar organelles, such as phagosomes, and will be the fluorophore of choice in this manuscript22,25. As a reference fluorophore, tetramethylrhodamine will be used, which is insensitive to pH (Figure 1E). Other fluorophores, such as pHrodo and cypHer5e may be substituted for fluorescein where the spectral properties of fluorescein do match other experimental variables. Some suggested reference fluorophores for pHrodo and cypHer5e are shown in Figure 1.
A second consideration is the method by which the two fluorophores will be targeted specifically to macropinosomes. Dextran of the size 70 kDa, which has a hydrodynamic radius of roughly 7 nm, does not stick non-specifically to cells and is incorporated into macropinosomes, but not clathrin-coated pits or caveolae, and therefore marks macropinosomes (Figure 2A and Figure 3A,B)16,24,26. In this protocol, fluorescein-labeled 70 kDa dextran and tetramethylrhodamine (TMR)-labeled 70 kDa dextran will be used as the pH-sensitive and reference probes, respectively.
In innate immune cells, macropinocytosis and phagocytosis represent the two major routes for the internalization of exogenous material for processing and subsequent presentation to cells of the adaptive immune response27. The careful and coordinated control of the redox chemistry of the lumen of phagosomes and macropinosomes is critical to the context-specific processing of exogenous material. Perhaps the most well-studied regulator of oxidative events in phagosomes is the NADPH oxidase, a large multi-subunit complex that produces large quantities of reactive oxygen species (ROS) within the lumen of phagosomes28. Indeed, its activity is central to appropriate antigen processing within phagosomes29,30. Yet, the activity of the NADPH oxidase on macropinosomal membranes has not been explored.
In this protocol, the H2DCFDA succinimidyl ester is used for measuring oxidative events within the macropinosome. This is a modified form of fluorescein (2&#39;,7&#39;-dichlorodihydrofluorescein diacetate), which is minimally fluorescent in its reduced form. Upon oxidation, its fluorescence emission increases significantly. It is, however, worth noting a significant caveat of H2DCFDA - as it is based on the fluorophore fluorescein, its fluorescence is also quenched in acidic compartments, and care must be taken to control for this variable when designing experiments28. Similar to the approach for measuring pH, the H2DCFDA succinimidyl ester will be covalently attached to 70 kDa dextran and TMR-labeled 70 kDa dextran will be used as the reference fluorophore (Figure 3A).
Fluorescent ovalbumin will be used to measure protein degradation within macropinosomes. The ovalbumin used here is densely labeled with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) FL dye that is self-quenched. Upon digestion, strongly fluorescent dye-labeled peptides are liberated. As ovalbumin cannot be easily conjugated to 70 kDa dextran, cells with TMR-labeled 70 kDa dextran and fluid-phase ovalbumin will be co-incubated. The TMR signal will be used to generate a macropinosome mask during post-imaging analysis, and the signal liberated from the digested ovalbumin will be measured within the mask (Figure 3B).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Black-walled 96 well plate</td>
			<td>PerkinElmer</td>
			<td>6005430</td>
			<td></td>
		</tr>
		<tr>
			<td>CypHer5e, NHS ester</td>
			<td>Cytiva</td>
			<td>PA15401</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-amino 70 kDa</td>
			<td>Invitrogen</td>
			<td>D1862</td>
			<td></td>
		</tr>
		<tr>
			<td>DQ-ovalbumin</td>
			<td>Invitrogen</td>
			<td>D12053</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-dextran 70 kDa</td>
			<td>Invitrogen</td>
			<td>D1823</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14287</td>
			<td></td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma Aldrich</td>
			<td>N7143</td>
			<td></td>
		</tr>
		<tr>
			<td>OxyBurst Green-SE</td>
			<td>Invitrogen</td>
			<td>D2935</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red, SE</td>
			<td>Invitrogen</td>
			<td>P36600</td>
			<td></td>
		</tr>
		<tr>
			<td>Raw264.7 cells</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>SP5 Confocal Microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-dextran 70 kDa</td>
			<td>Invitrogen</td>
			<td>D1819</td>
			<td></td>
		</tr>
		<tr>
			<td>u-Dish</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the ph, redox chemistries, and degradative capacity of macropinosomes using dual-fluorophore ratiometric microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

When measuring macropinosome pH, there is a period of time for which the dynamics of acidification cannot be measured. This period corresponds to the dextran loading phase (gray box in Figure 2C and Figure 3C,D) and will vary depending on the cell type used. The length of the loading phase will vary with (i) the macropinocytic activity of the cells and (ii) the sensitivity of the instrument used. It is recommended to adjust this period at the st...

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

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

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.

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