pH Indicator Conjugates—Section 20.4

This section includes our selection of pH indicators conjugated to dextrans and lipids, as well as our chemically reactive pH indicators for preparing new pH-sensitive conjugates. The pH indicator conjugates described below include both those useful at near-neutral pH and those useful in acidic environments.

The pH-sensitive properties of the pH indicators described in Probes Useful at Near-Neutral pH—Section 20.2 and Probes Useful at Acidic pH—Section 20.3 are usually not significantly affected upon conjugation to dextrans. However, coupling of pH indicators to these relatively inert polysaccharides changes several other properties of the dyes:

• Conjugates have high water solubility and therefore must be loaded into cells by microinjection, whole-cell patch-clamping, endocytosis or liposome fusion or by using the Influx pinocytic cell loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8).
• Once loaded, dextrans are retained in viable cells for long periods and (at least those dextrans with average molecular weights above 3000) will not pass through gap junctions.
• Attachment to a dextran significantly decreases the likelihood that the indicator will become compartmentalized, thereby avoiding a substantial problem associated with cell-permeant acetoxymethyl (AM) ester derivatives.

The properties of some of the most useful pH indicator dextrans available from Molecular Probes are listed in Molecular Probes' pH indicator dextrans, in order of decreasing pKa—Table 20.3 in approximate order of decreasing pK_a value. Our numerous labeled dextrans are discussed in Fluorescent and Biotinylated Dextrans—Section 14.5 and listed in Molecular Probes' selection of dextran conjugates—Table 14.4.

pHrodo Dextran

The 10,000 MW pHrodo dextran (P10361) is a superior alternative to other fluorescent dextran conjugates (e.g., BCECF and tetramethylrhodamine) for live-cell imaging of endocytosis, the process whereby the plasma membrane buds to form membrane bound vesicles, endosomes, which are then trafficked to various destinations within the cell. Endocytosis underlies several fundamental cellular processes including receptor desensitization, nutrient acquisition, and antigen presentation. Furthermore, new roles for endocytosis such as cytokinesis mediation, cell polarization, and cell migration are continuing to be identified.1 pHrodo dextran possesses a pH-sensitive fluorescence emission that increases in intensity with increasing acidity (Figure 1). pHrodo dextran is essentially dark in the extracellular environment; however, upon internalization, the acidic environment of the endosomes elicits a bright, red-fluorescent signal from this dextran conjugate. The minimal fluorescent signal from the pHrodo dextran conjugate at neutral pH prevents the detection of non-internalized and nonspecifically bound conjugates, and eliminates the need for quenching reagents and extra wash steps, thus providing a simplefluorescent assay for endocytotic activity. pHrodo dextran can be used to study or monitor endocytosis on a variety of platforms including fluorescence microscopy, flow cytometry, and automated imaging and analysis (also known as high-content imaging or HCS) (Figure 2). pHrodo dextran’s excitation and emission maxima of 560 and 585 nm, respectively, facilitates multiplexing with other fluorophores including blue, green, and far-red fluorescent probes. Localization of pHrodo dextran can be established using Organelle Lights Endosomes-GFP or Lysosomes- GFP reagents. While pHrodo dextran is optimally excited at approximately 560 nm, it is also readily excited by the 488 nm argon-ion laser installed on most flow cytometers, microscopes, and microplate readers.

Figure x.xx pHrodo dextran.

BCECF and SNARF Indicator Dextrans for Measuring Near-Neutral pH

Our 10,000 MW and 70,000 MW BCECF dextrans (D1878, D1880) are important dual-excitation pH indicator conjugates for pH measurements near pH 7.0. BCECF dextran–labeled Swiss 3T3 cells have been shown to produce much more stable fluorescent signals, reduced probe compartmentalization and 10-fold greater resistance to light-induced damage when compared with BCECF AM–labeled cells. The 10,000 MW BCECF dextran (D1878) has been used to monitor intracellular pH increases during developmental processes and to measure pH in submucosal gland secretions from human lung tissues. Cytoplasmic Ca²⁺/H⁺ buffering in green algae has been investigated using BCECF dextran in combination with fura dextran (F3029, Fluorescent Ca2+ Indicator Conjugates—Section 19.4).

A dextran conjugate of the carboxy SNARF-1 pH indicator (D3303, D3304) has been microinjected into rhizoid cells of the alga Pelvetia fastigata and used with ratiometric imaging to measure pH gradients associated with polar tip growth. SNARF-1 dextran has also been used to detect cytosolic alkalinization associated with multidrug transporter activity and to investigate pH regulation of connexin 43 channels. SNARF dextran conjugates have been scrape-loaded into the cytosol of MDF-7/ADR cells. It was found that the 70,000 MW SNARF dextran conjugate remained exclusively cytosolic, whereas the 10,000 MW conjugate reported the pH of both cytosolic and nuclear compartments.

Indicator Dextrans for Measuring Acidic pH

Although the fluorescein, BCECF and SNARF dextrans are intended for pH measurements between pH ~6 and 8, these dextrans are also useful for detecting uptake into acidic organelles, such as occurs during endocytosis. In particular, when these indicator dextrans enter moderately acidic compartments (pH <5.5):

• Fluorescence of the fluorescein and BCECF dextrans is strongly quenched (Probes Useful at Near-Neutral pH—Section 20.2).
• The 520/570 nm emission intensity ratio of the double-labeled fluorescein–tetramethylrhodamine dextran (D1951) decreases (Figure 20.28).
• The 580/640 nm emission ratio of the SNARF-1 dextrans increases (Probes Useful at Near-Neutral pH—Section 20.2).

The fluorescein, BCECF and SNARF dextrans are useful for detecting translocation into compartments that have an acidic pH; however, the relative insensitivity of their fluorescence below pH ~6 limits quantitative pH estimation. The lower pK_a values of the Oregon Green 488 and Oregon Green 514 dextran conjugates (Molecular Probes' pH indicator dextrans, in order of decreasing pKa—Table 20.3) make them more suitable indicators for estimating the pH of relatively acidic lysosomal environments. Moreover, the shift in their excitation spectra in acidic media permits ratiometric pH measurements.

We have also developed a 10,000 MW dextran conjugate of the LysoSensor Yellow/Blue dye (L22460), which can be used to quickly and accurately measure the pH of lysosomes. As this labeled dextran is taken up by the cells and moves through the endocytic pathway, the fluorescence of the LysoSensor probe changes from blue fluorescent in the near-neutral endosomes to yellow fluorescent in the acidic lysosomes. The greatest change in fluorescence emission occurs near the pK_a of the dye at pH ~4.2. The pH in lysosomes can be measured with LysoSensor Yellow/Blue dextran using fluorescence microscopy () or flow cytometry.

Figure 20.28 The excitation spectra of double-labeled fluorescein–tetramethylrhodamine dextran (D1951), which contains pH-dependent (fluorescein) and pH-independent (tetramethylrhodamine) dyes.

Lipophilic Fluorescein

Measurement of the pH adjacent to membrane surfaces is often complicated by electrostatic charge and solvation effects on the pK_a of surface-bound indicators. The pK_a of membrane-intercalated fluorescein DHPE (F362, ) is ~6.2, quite close to that of free fluorescein. Researchers have used the pH-dependent fluorescence of fluorescein DHPE to measure lateral proton conduction along lipid monolayers. This fluorescein-labeled phospholipid has also been used to follow proton translocation from internal compartments in phospholipid vesicles. For more acidic environments, Oregon Green 488 DHPE (O12650, ) has potentially similar applications. Other related lipophilic fluorescein derivatives, including 5-dodecanoylaminofluorescein (D109) and 5-hexadecanoylaminofluorescein (H110), are described in Other Nonpolar and Amphiphilic Probes—Section 13.5.

Lipophilic Coumarin

4-Heptadecyl-7-hydroxycoumarin (H22730, ) is an alkyl derivative of the pH-sensitive blue-fluorescent 7-hydroxycoumarin (umbelliferone) fluorophore. As with other lipophilic coumarins, 4-heptadecyl-7-hydroxycoumarin is primarily used as a probe of membrane surfaces. The pK_a of 4-heptadecyl-7-hydroxycoumarin varies from 6.35 in the cationic detergent CTAB to 11.15 in the anionic detergent sodium dodecyl sulfate (SDS), as measured by its fluorescence response. The pK_a values of lipophilic pH indicators are strongly dependent on the ionic composition of the membrane surface, making them sensitive probes of membrane-surface electrostatic potential. 4-Heptadecyl-7-hydroxycoumarin has been used to measure pH differences at membrane interfaces in isolated plasma membranes of normal and multidrug-resistant murine leukemia cells and to characterize interactions of cationic lipids with DNA.

Amine-Reactive pHrodo Dye for Measuring Acidic pH

pHrodo dye is a novel, fluorogenic dye that dramatically increases in fluorescence as the pH of its surroundings becomes more acidic. This amine-reactive succinimidyl ester form of the dye (P36600) has a pKa of ~7.3 in solution, which shifts to about ~6.5 upon conjugation to the K-12 strain of Escherichia coli (Figure 20.32) pHrodo dye is extremely sensitive to its local environment, therefore the pH response in your system will need to be determined empirically.

To study endocytosis in a high-throughput format, we have used pHrodo conjugates to create dose response curves for the inhibition of endocytosis by dynasore, a dynamin-specific inhibitor. pHrodo succinimidyl ester was conjugated to an amine-derivatized 10,000 MW dextran, and cells were treated with serial dilutions of dynasore to create dose response curves for endocytosis inhibition (Figure 20.33). The loss of pHrodo dextran accumulation in punctuate structures within the cell was observed as a function of dynasore concentration.

Figure 20.32 The fluorescence emission spectra of pHrodo dye–labeled Escherichia coli were measured in a series of 50 mM potassium phosphate buffers ranging in pH from 4 to 10. The E. coli were at a concentration of 0.1 mg/mL, and the readings were made on a Hitachi F4500 fluorometer, using an excitation wavelength of 532 nm.

Figure 20.33 Tracking endocytosis inhibition with pHrodo dextran conjugates. HeLa cells were plated in 96-well format and treated with dynasore for 3 hours at 37°C prior to the pHrodo assay. Then, 40 µg/mL pHrodo dextran synthesized from pHrodo succinimidyl ester (P36600) and amine-reactive 10,000 MW dextran (D1860) was incubated for 30 min at 37°C. Cells were stained with HCS NuclearMask Blue stain (H10325) for 10 min to reveal total cell number and demarcation for image analysis. Images were acquired on the BD Pathway 855 High-Content Bioimager (BD Biosciences).

Other Amine-Reactive Dyes

Many of the pH indicators described in Probes Useful at Near-Neutral pH—Section 20.2 and Probes Useful at Acidic pH—Section 20.3 can be conjugated to biological molecules in order to generate pH-sensitive tracers. The resulting conjugates are useful for following endocytosis, phagocytosis, organelle trafficking and other processes, as described in Probes for Following Receptor Binding, Endocytosis and Exocytosis—Section 16.1. For example, the pH sensitivity of fluorescein-labeled transferrin (T2871, Probes for Following Receptor Binding, Endocytosis and Exocytosis—Section 16.1) has frequently been exploited to detect pH changes associated with the endocytic processing of this important iron-transporting glycoprotein.

For these types of applications, fluorescein conjugates are less than optimal because they have little sensitivity in the pH range below pH 5.5. The response range can be extended using conjugates of our Oregon Green 488 dye, which has a much lower pK_a than fluorescein (4.7 compared with 6.4, Figure 1.12) but essentially identical spectra. Our collaborators, Dr. Elizabeth Simons and her co-workers, have labeled fungi (Cryptococcus neoformans) with FITC (F143, F1906, F1907; Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) or Oregon Green 488 isothiocyanate (O6080, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) and used them to study the influence of phagosomal pH on the fungicidal and fungistatic activity of human monocyte–derived macrophages. At pH levels evoked by phagocytosis of live and heat-killed fungi (pH 4.7–5.7), standard curves of the 498/450 nm fluorescence excitation ratio as a function pH (Figure 20.31) illustrate the greater sensitivity of the Oregon Green 488 conjugates. Vergne and co-workers have used zymosan (heat-killed yeast) double-labeled with the succinimidyl esters of Oregon Green 488 carboxylic acid (O6147) and carboxytetramethylrhodamine (C1171, Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6) to measure phagosomal pH in J774 macrophages using dual-emission (530/585 nm) flow cytometry; they were able to estimate pH values as low as 4.0.

Many of the reagents or methods required to prepare conjugates have been described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1. The most common method of producing a useful conjugate is the reaction of amines with succinimidyl esters or isothiocyanates of the pH indicator. Examples of amine-reactive pH indicators include the succinimidyl esters of the carboxy SNARF-1 and carboxynaphthofluorescein dyes (S22801, C653). The succinimidyl ester of the chlorinated fluorescein derivative 6-JOE (pK_a ~11.5, C6171MP; Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) can be used to prepare conjugates that are responsive to alkaline pH levels. When the amine-reactive pH indicator is not available, sulfosuccinimidyl esters can generally be prepared in situ simply by dissolving the carboxylic acid dye in a buffer that contains N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHSS, H2249; EDAC, E2247; Derivatization Reagents for Carboxylic Acids and Glutamine—Section 3.3). Addition of NHSS to the buffer has been shown to enhance the yield of carbodiimide-mediated conjugations. Suitable amine-reactive pH indicators or dyes that can be made reactive using EDAC/NHSS are listed in Reactive pH indicator dyes—Table 20.4.

Figure 1.12 Comparison of pH-dependent fluorescence of the Oregon Green 488 (), carboxyfluorescein () and Alexa Fluor 488 () fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm.

Figure 20.31 Calibration curves for intraphagosomal pH measurements using fungi (Cryptococcus neoformans) labeled with Oregon Green 488 isothiocyanate (O6080) or fluorescein isothiocyanate (FITC; F143, F1906, F1907). Human monocyte–derived macrophages laden with phagocytosed C. neoformans were exposed to pH-controlled buffers in the presence of the K+/H+ ionophore nigericin (N1495). The 498/450 nm fluorescence excitation ratios corresponding to different pH levels were measured in a spectrofluorometer. The data were provided by Elizabeth Simons, Boston University.