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 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 are listed in Molecular Probes pH indicator dextrans, in order of decreasing pKa—Table 20.3 in approximate order of decreasing pKa value. Our numerous labeled dextrans are discussed in Fluorescent and Biotinylated Dextrans—Section 14.5 and listed in Molecular Probes dextran conjugates—Table 14.4.
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 (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). pHrodo dextran possesses a pH-sensitive fluorescence emission that increases in intensity with increasing acidity (Figure 20.4.1). pHrodo dextran is essentially nonfluorescent 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 noninternalized and nonspecifically bound conjugates and eliminates the need for quenching reagents and extra wash steps, thus providing a simple fluorescent assay for endocytic activity (Figure 20.4.3). 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). pHrodo dextran’s excitation and emission maxima of 560 and 585 nm, respectively, facilitate multiplexing with other fluorophores including Alexa Fluor 488 and Alexa Fluor 647 dyes as well as green-fluorescent protein (GFP).
Figure 20.4.1 The pH response profile of pHrodo dextran (P10361) monitored at excitation/emission wavelengths of 545/590 nm in a fluorescence microplate reader. Citrate, MOPS and borate buffers were used to span the pH range from 2.5 to 10.
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.
Oregon Green and LysoSensor Yellow/Blue Dextrans
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 20.4.2).
- 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 pKa 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 estimate 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 pKa 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 20.4.2 The excitation spectra of double-labeled fluorescein–tetramethylrhodamine dextran (D1951), which contains pH-dependent (fluorescein) and pH-independent (tetramethylrhodamine) dyes.
Measurement of the pH adjacent to membrane surfaces is often complicated by electrostatic charge and solvation effects on the pKa of surface-bound indicators. The pKa 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.
Amine-Reactive pHrodo pH Indicator
pHrodo dye is an aminorhodamine pH indicator that increases in fluorescence as the pH of its surroundings becomes more acidic (Figure 20.4.1). The amine-reactive succinimidyl ester form of the dye (P36600) provides access to a wide variety of user-defined bioconjugates by following the detailed protocols provided in the accompanying product information sheet. pHrodo dye is extremely sensitive to its local environment; therefore the pH response of each bioconjugate must be individually determined. pHrodo succinimidyl ester has been used to label dexamethansone-treated thymocytes for flow cytometric analysis of phagocytosis by splenic or peritoneal macrophages and for live-cell confocal imaging of antigen transfer from human B lymphocytes to macrophages.
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 20.4.3). The loss of pHrodo dextran accumulation in punctuate structures within the cell was observed as a function of dynasore concentration.
Figure 20.4.3 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 10,000 MW aminodextran (D1860) was incubated for 30 minutes at 37°C. Cells were stained with HCS NuclearMask Blue stain (H10325) for 10 minutes 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 and Phagocytosis—Section 16.1. For example, the pH sensitivity of fluorescein-labeled transferrin (T2871, Probes for Following Receptor Binding and Phagocytosis—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 pKa than fluorescein (4.7 compared with 6.4, Figure 20.4.4) 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 20.4.5) 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 ester or isothiocyanate derivatives 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 (pKa ~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 Carboxamides—Section 3.4); this method was used to activate SNARF-4F carboxylic acid (S23920, Probes Useful at Near-Neutral pH—Section 20.2) for labeling 5'-amine–modified oligonucleotides. 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 20.4.4 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 20.4.5 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.
| Acidic Solution || Basic Solution |
|C653||573.51||F,D,L||DMF, DMSO||515||10,000||565||pH 6||602||42,000||672||pH 10||7.6||1, 2|
|D1821||see Notes||F,L||H2O||473||ND||514||pH 5||490||ND||513||pH 9||6.4||1, 3, 4, 5|
|D1823||see Notes||F,L||H2O||473||ND||514||pH 5||490||ND||514||pH 9||6.4||1, 3, 4, 5|
|D1844||see Notes||F,L||H2O||473||ND||514||pH 5||490||ND||514||pH 9||6.4||1, 3, 4, 5|
|D1878||see Notes||F,L||H2O||482||ND||520||pH 5||503||ND||528||pH 9||7.0||1, 3, 4, 5|
|D1880||see Notes||F,L||H2O||482||ND||520||pH 5||503||ND||528||pH 9||7.0||1, 3, 4, 5|
|D1951||see Notes||F,L||H2O||see Notes||see Notes||ND||1, 3, 4, 6|
|D3303||see Notes||F,L||H2O||548||ND||587||pH 6||576||ND||635||pH 10||7.5||1, 3, 4, 5|
|D3304||see Notes||F,L||H2O||548||ND||587||pH 6||576||ND||635||pH 10||7.5||1, 3, 4, 5|
|D3305||see Notes||F,L||H2O||473||ND||514||pH 5||490||ND||514||pH 9||6.4||1, 3, 4, 5|
|D7170||see Notes||F,L||H2O||478||ND||518||pH 3||492||ND||518||pH 9||4.7||1, 3, 4, 5|
|D7172||see Notes||F,L||H2O||478||ND||518||pH 3||492||ND||518||pH 9||4.7||1, 3, 4, 5|
|D7176||see Notes||F,L||H2O||489||ND||526||pH 3||506||ND||526||pH 9||4.7||1, 3, 4, 5|
|F362||1182.54||FF,D,L||see Notes||476||32,000||519||MeOH/H+||496||88,000||519||MeOH/OH–||6.2||7, 8|
|L22460||see Notes||F,D,L||H2O||384||ND||540||pH 3||329||ND||440||pH 7.5||4.2||1, 3, 4, 5|
|O6139||609.43||F,D,L||DMF, DMSO||489||26,000||526||pH 3||506||85,000||526||pH 9||4.7||2|
|O6147||509.38||F,D,L||DMF, DMSO||480||24,000||521||pH 3||495||76,000||521||pH 9||4.7||2|
|O6149||509.38||F,D,L||DMF, DMSO||480||26,000||516||pH 3||496||82,000||516||pH 9||4.7||2|
|O12650||1086.25||FF,D,L||see Notes||485||26,000||526||MeOH/H+||501||85,000||526||MeOH/OH–||ND||4, 7, 8|
|P10361||see Notes||F,L||H2O||560||ND||587||pH 2||553||ND||587||pH 9||see Notes||3, 4, 9|
|P36600||~650||F,D,L||DMSO||560||95,000||587||pH 2||553||90,000||587||pH 9||see Notes||9|
|1. Spectra are in aqueous buffers adjusted to >1 pH unit above and >1 pH unit below the pKa.|
|2. Spectral data for this product represents the unreacted succinimidyl ester. The pKa value and the spectral data for acidic solutions have been estimated based on the spectra of the parent carboxylic acid.|
|3. The molecular weight is nominally as specified in the product name but may have a broad distribution.|
|4. ND = not determined.|
|5. Abs, Em and pKa values listed for this dextran conjugate are those obtained for the free dye. Values for actual conjugates are typically very similar, with slight variations between different production lots.|
|6. These conjugates contain both pH-sensitive fluorescein (Abs = 495, Em = 520 nm) and pH-insensitive tetramethylrhodamine (Abs = 555 nm, Em = 575 nm) fluorophores.|
|7. The pKa values of lipophilic pH indicators may vary considerably depending on the electrostatic properties of membrane surfaces. The pKa values listed are for electrostatically neutral liposomes or micelles. Spectra are in MeOH containing a trace of HCl (MeOH/H+) or a trace of KOH (MeOH/OH–).|
|8. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonication to aid dispersion if necessary. Labeling of cells with fluorescent phospholipids can be enhanced by addition of cyclodextrins during incubation.|
|9. pHrodo succinimidyl ester (P36600) exhibits a complex pH titration profile. Decreasing pH (from pH 9 to pH 2) produces a continuous (but nonlinear) fluorescence increase. This pH response profile typically changes upon conjugation of the dye to proteins and other biomolecules.|
|10. S22801 is converted to fluorescent products with spectra similar to C1270 after acetate hydrolysis.|