- Fluorescent Fatty Acid Analogs
- ADIFAB Fatty Acid Indicator
- Phospholipids with BODIPY Dye–Labeled Acyl Chains
- Phospholipid with DPH-Labeled Acyl Chain
- Phospholipids with NBD-Labeled Acyl Chains
- Phospholipids with Pyrene-Labeled Acyl Chains
- Phospholipids with a Fluorescent or Biotinylated Head Group
- LipidTOX Phospholipid and Neutral Lipid Stains for High-Content Screening
- Data Table
- Ordering Information
The probes in this section and in Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3 bear some structural resemblance to natural lipids. Included in this section are fluorescent fatty acid analogs, as well as phospholipids wherein one or both fatty acid esters are replaced by fluorescent fatty acid esters. The fluorophores in these probes tend to remain buried in the hydrophobic interior of lipid bilayer membranes. In this location, they are sensitive to membrane properties such as lipid fluidity, lateral domain formation and structural perturbation by proteins, drugs and other additives. Also included in this section are several head group–modified phospholipid analogs incorporating a fluorophore or biotin (Phospholipids with labeled head groups—Table 13.1).
Sphingolipids, steroids and lipopolysaccharides are discussed in Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3. Important applications of the fluorescent phosphatidylinositol derivatives as probes for signal transduction and various fluorescent phospholipids as phospholipase substrates are further described in Probes for Lipid Metabolism and Signaling—Section 17.4. A review of fluorescent lipid probes and their use in biological and biophysical research has been published.
Our fluorescent fatty acid analogs have a fluorophore linked within the fatty acid chain or, more commonly, at the terminal (omega) carbon atom that is furthest from the carboxylate moiety. Although fluorescent fatty acid analogs are sometimes used as direct probes for membranes and liposomes, their most common applications have been for synthesis of fluorescent phospholipids and for metabolic incorporation by live cells. Our fluorescent fatty acids currently include derivatives based on the BODIPY, nitrobenzoxadiazole (NBD), pyrene and dansyl fluorophores, as well as the naturally fluorescent polyunsaturated fatty acid, cis-parinaric acid.
BODIPY Fatty Acids
BODIPY fatty acids are, by far, the most fluorescent fatty acid analogs that we have available. The lack of ionic charge on the BODIPY fluorophore is unusual for long-wavelength fluorescent dyes and results in exclusive localization of the fluorophore within the membrane (Figure 13.2.1F). BODIPY derivatives typically have extinction coefficients greater than 90,000 cm-1M-1 with absorption maxima beyond 500 nm. A useful spectroscopic property of BODIPY dyes is the concentration-dependent formation of excited-state dimers ("excimers") with red-shifted emission. We have observed this phenomenon particularly with our green-fluorescent BODIPY fatty acid derivatives, which undergo a considerable red shift in their emission when metabolically incorporated into lipophilic products (). Pyrene fatty acids (see below) also exhibit excimer formation but their emission is at much shorter wavelengths than that of the BODIPY dyes and they are therefore less suitable for the study of live cells. The fluorophores in our current selection of BODIPY fatty acids and their approximate absorption/emission maxima (in nm) are:
BODIPY fatty acids are synthetic precursors to a wide variety of fluorescent phospholipids (described below), as well as several important sphingolipid probes described in Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3. Some BODIPY fatty acids are readily metabolized by live cells to phospholipids, di- and triacylglycerols, cholesteryl esters and other natural lipids. Analysis of cellular lipid extracts by HPLC has shown that glycerophosphocholines constitute more than 90% of the products of biosynthetic incorporation of BODIPY 500/510 dodecanoic acid (D3823) by BHK cells.
The three BODIPY 500/510 probes form a unique series in which the green-fluorescent fluorophore is located within the fatty acid chain at different distances from the terminal carboxylate group. The overall length of the probe is constant and, including the fluorophore, is about equivalent to that of an 18-carbon fatty acid (Figure 13.2.2).
BODIPY 581/591 undecanoic acid (D3861) is particularly useful for detecting reactive oxygen species in cells and membranes. Oxidation of the polyunsaturated butadienyl portion of the BODIPY 581/591 dye () truncates the conjugated π-electron system, resulting in a shift of the fluorescence emission peak from ~590 nm to ~510 nm. This oxidation response mechanism is similar to that of the naturally occurring polyunsaturated fatty acid cis-parinaric acid (see below). In comparison to cis-parinaric acid, advantages of BODIPY 581/591 undecanoic acid include:
- Long-wavelength excitation, compatible with confocal laser-scanning microscopes and flow cytometers
- Avoidance of photooxidation effects induced by ultraviolet excitation
- Less interference by colored oxidant and antioxidant additives when detecting probe fluorescence
- Greater resistance to spontaneous oxidation
- Red-to-green fluorescence shift, allowing the use of fluorescence ratio detection methods
Figure 13.2.1 Location and orientation of representative fluorescent membrane probes in a phospholipid bilayer: A) DPH (D202), B) NBD-C6-HPC (N3786), C) bis-pyrene-PC (B3782), D) DiI (D282), E) cis-parinaric acid (P36005), F) BODIPY 500/510 C4, C9 (B3824), G) N-Rh-PE (L1392), H) DiA (D3883) and I) C12-fluorescein (D109).
Figure 13.2.2 Structural representations showing the positional shift of the fluorophore with respect to the terminal carboxyl group in a homologous series of BODIPY 500/510 fatty acids (D3823, B3824, D3825).
NBD Fatty Acids
Fluorescence of the nitrobenzoxadiazole (NBD) fluorophore is highly sensitive to its environment. Although it is moderately fluorescent in aprotic solvents, in aqueous solvents it is almost nonfluorescent. The NBD fluorophore is moderately polar and both its homologous 6-carbon and 12-carbon fatty acid analogs (N316, ; N678) and the phospholipids derived from these probes (N3786, N3787) tend to sense the lipid–water interface region of membranes instead of the hydrophobic interior (Figure 13.2.1B). The environmental sensitivity of NBD fatty acids can be usefully exploited to probe the ligand-binding sites of fatty acid and sterol carrier proteins. NBD fatty acids are not well metabolized by live cells.
Pyrene Fatty Acids
The hydrophobic pyrene fluorophore is readily accommodated within the membrane. ω-Pyrene derivatives of longer-chain fatty acids () were first described by Galla and Sackmann in 1975. We offer pyrene derivatives of the 4-, 10-, 12- and 16-carbon fatty acids (P1903MP, P31, P96, P243, respectively). Pyrenebutanoic acid—frequently called pyrenebutyric acid (P1903MP)—has rarely been used as a membrane probe; however, its conjugates have exceptionally long excited-state lifetimes (τ >100 nanoseconds) and are consequently useful for time-resolved fluorescence immunoassays and nucleic acid detection. The long excited-state lifetime of pyrenebutyric acid also makes it useful as a probe for oxygen in cells and lipid vesicles.
Pyrene derivatives form excited-state dimers (excimers) with red-shifted fluorescence emission (Figure 13.2.3). Pyrene excimers can even form when two pyrenes are tethered by a short trimethine spacer, as in 1,3-bis-(1-pyrenyl)propane (B311, Other Nonpolar and Amphiphilic Probes—Section 13.5). Pyrene excimer formation is commonly exploited for assaying membrane fusion (Lipid-Mixing Assays of Membrane Fusion—Note 13.1) and for detecting lipid domain formation. Pyrene fatty acids are metabolically incorporated into phospholipids, di- and tri-acylglycerols and cholesteryl esters by live cells. Other uses of pyrene fatty acids include:
Figure 13.2.3 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.
Dansyl Undecanoic Acid
Dansyl undecanoic acid (DAUDA, D94; ) incorporates a polar, environment-sensitive dansyl fluorophore that preferentially locates in the polar headgroup region of lipid bilayer membranes. DAUDA exhibits a 60-fold fluorescence enhancement and a large emission spectral shift to shorter wavelengths on binding to certain proteins. This property has been exploited to analyze fatty acid–binding proteins and also to develop a fluorometric phospholipase A2 assay (Probes for Lipid Metabolism and Signaling—Section 17.4) based on competitive fatty acid displacement.
The naturally occurring polyunsaturated fatty acid cis-parinaric acid (P36005, ) was initially developed as a membrane probe by Hudson and co-workers and published in 1975. cis-Parinaric acid is the closest structural analog of intrinsic membrane lipids among currently available fluorescent probes (Figure 13.2.1E). The chemical and physical properties of cis-parinaric acid have been well characterized. The lowest absorption band of cis-parinaric acid has two main peaks around 300 nm and 320 nm, with a high extinction coefficient. cis-Parinaric acid offers several experimentally advantageous optical properties, including a very large fluorescence Stokes shift (~100 nm) and an almost complete lack of fluorescence in water. In addition, the fluorescence decay lifetime of cis-parinaric acid varies from 1 to ~40 nanoseconds, depending on the molecular packing density in phospholipid bilayers. Consequently, minutely detailed information on lipid-bilayer dynamics can be obtained.
Selected applications of cis-parinaric acid include:
- Measurement of peroxidation in lipoproteins and the relationship of peroxidation to cytotoxicity and apoptosis
- Evaluation of antioxidants
- Detection of lipoproteins following chromatographic separation and structural characterization of lipoproteins
- Detection of lipid–protein interactions and lipid clustering
- High-affinity binding to a hydrophobic pocket between the heavy chain of myosin subfragment-1 and its essential light chain
- Investigation of the mechanism of fatty acid–binding proteins and phospholipid-transfer proteins
The extensive unsaturation of cis-parinaric acid makes it quite susceptible to oxidation. Consequently, we offer cis-parinaric acid in a 10 mL unit size of a 3 mM solution in deoxygenated ethanol (P36005); if stored protected from light under an inert argon atmosphere at -20°C, this stock solution should be stable for at least six months. During experiments, we strongly advise handling cis-parinaric acid samples under inert gas and preparing solutions using degassed buffers and solvents. cis-Parinaric acid is also somewhat photolabile, undergoing photodimerization under intense illumination, resulting in loss of fluorescence.
Fatty acid–binding proteins are small cytosolic proteins found in a variety of mammalian tissues, and studies of their physiological function frequently involve fluorescent fatty acid probes. To facilitate these studies, we offer ADIFAB (A3880), a dual-wavelength fluorescent indicator of free fatty acids (Figure 13.2.4, Figure 13.2.5). ADIFAB is a conjugate of I-FABP, a rat intestinal fatty acid–binding protein with a low molecular weight (15,000 daltons) and a high binding affinity for free fatty acids, and the polarity-sensitive acrylodan fluorophore (A433, Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3). It is designed to provide quantitative monitoring of free fatty acids without resorting to separative biochemical methods. With appropriate precautions, which are described in the product information sheet accompanying this product, ADIFAB can be used to determine free fatty acid concentrations between 1 nM and >20 µM.
Figure 13.2.4 Ribbon representation of the ADIFAB free fatty acid indicator (A3880). In the left-hand image, the fatty acid binding site of intestinal fatty acid–binding protein (yellow) is occupied by a covalently attached acrylodan fluorophore (blue). In the right-hand image, a fatty acid molecule (gray) binds to the protein, displacing the fluorophore (green) and producing a shift of its fluorescence emission spectrum. Image contributed by Alan Kleinfeld, FFA Sciences LLC, San Diego.
Figure 13.2.5 The free fatty acid–dependent spectral shift of ADIFAB (A3880). Spectra shown represent 0.2 µM ADIFAB in pH 8.0 buffer with (+OA) and without (–OA) addition of 4.7 µM cis-9-octadecenoic (oleic) acid (OA). The ratio of fluorescence emission intensities at 505 nm and 432 nm can be quantitatively related to free fatty acid concentrations.
We offer several glycerophospholipid analogs labeled with a single green-fluorescent BODIPY 500/510 or a BODIPY FL fluorophore or red-orange–fluorescent BODIPY 530/550 fluorophore on the sn-2 acyl chain, including:
In addition, we prepare a glycerophosphocholine analog with a single nonhydrolyzable ether-linked BODIPY FL fluorophore on the sn-1 position (D3771, ), as well as several doubly labeled glycerophospholipids. These doubly labeled glycerophospholipids, which are discussed in greater detail in Probes for Lipid Metabolism and Signaling—Section 17.4, are designed primarily for detection of phospholipase A1 and phospholipase A2 and include:
- Glycerophosphoethanolamine with a BODIPY FL dye–labeled sn-1 acyl chain and a dinitrophenyl quencher–modified headgroup (PED-A1, A10070; )
- Glycerophosphoethanolamine with a BODIPY FL dye–labeled sn-2 acyl chain and a dinitrophenyl quencher–modified headgroup (PED6, D23739; Figure 13.2.9)
- Glycerophosphocholine with two BODIPY FL dye–labeled acyl chains (bis-BODIPY FL C11-PC, B7701; Figure 13.2.9)
- Glycerophosphocholine with a BODIPY 558/568 dye–labeled sn-1 alkyl chain and a BODIPY FL dye–labeled sn-2 acylchain (Red/Green BODIPY PC-A2, A10072; )
The spectral properties of BODIPY FL dye–labeled phospholipids are summarized in Spectral properties of some lipid probes—Table 13.2. Unlike the nitrobenzoxadiazole (NBD) fluorophore, the BODIPY FL and BODIPY 500/510 fluorophores are intrinsically lipophilic and readily localize in the membrane's interior. The fluorophore is completely inaccessible to the membrane-impermeant anti–BODIPY FL antibody (A5770, Anti-Dye and Anti-Hapten Antibodies—Section 7.4), which also recognizes the BODIPY 500/510 derivative. As shown in Figure 13.2.6, the emission spectrum of the BODIPY 500/510 fluorophore is much narrower than that of the NBD fluorophore. Because both the extinction coefficient of the BODIPY 500/510 fluorophore and its quantum yield in a lipophilic environment (EC ~90,000 cm-1M-1 and QY ~0.9) are much higher than those of the NBD fluorophore (EC ~20,000 cm-1M-1 and QY ~0.3), much less BODIPY 500/510 dye–labeled phospholipid is required for labeling membranes.
Incorporation of high molar ratios (>10 mole %) of the BODIPY 500/510 dye–labeled phospholipids into membranes results in a dramatic spectral shift of the fluorescence emission spectrum to longer wavelengths (Figure 13.2.7). We have also observed this spectral shift in the Golgi of live cells that have been labeled with our BODIPY dye–labeled ceramides (Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4) and with BODIPY fatty acids that have been metabolically incorporated by cells (). In fluorescence resonance energy transfer (FRET) measurements, the green-fluorescent BODIPY 500/510 dye is an excellent donor to longer-wavelength BODIPY probes (Figure 13.2.8) and acceptor from dansyl-, DPH- or pyrene-labeled phospholipids. These probe combinations offer several alternatives to the widely used NBD–rhodamine fluorophore pair for researchers using FRET techniques to study lipid transfer and membrane fusion.
Figure 13.2.6 Fluorescence spectra (excitation at 475 nm) of β-BODIPY 500/510 C12-HPC (blue line peak at 516 nm, D3793) and NBD C12-HPC (red line peak at 545 nm, N3787) incorporated in DOPC (dioctadecenoylglycerophosphocholine) liposomes at molar ratios of 1:400 mole:mole (labeled:unlabeled PC). The integrated intensities of the spectra are proportional to the relative fluorescence quantum yields of the two probes.
Figure 13.2.7 A) Fluorescence spectrum of β-C8-BODIPY 500/510 C5-HPC (D3795) incorporated in DOPC (dioctadecenoylphosphocholine) liposomes at 1:100 mole:mole (labeled:unlabeled PC). B) Fluorescence spectra at high molar incorporation levels: 1:10 mole:mole and 1:5 mole:mole.
Figure 13.2.8 Fluorescence resonance energy transfer from β-BODIPY 500/510 C12-HPC (peak at 516 nm, D3793) to BODIPY 558/568 C12 (peak at 572 nm, D3835) in DOPC (dioctadecenoylglycerophosphocholine) lipid bilayers using 475 nm excitation. Ratio of acceptors to donors is: 1) 0; 2) 0.2; 3) 0.4; 4) 0.8; and 5) 2.0.
Once cells are labeled with a BODIPY phospholipid, the probe shows little tendency to spontaneously transfer between cells. Consequently, BODIPY dye–labeled phospholipids have been used in a number of studies of cell membrane structure and properties:
- Despite their good photostability, BODIPY lipids are useful for fluorescence recovery after photobleaching (FRAP) measurements of lipid diffusion.
- Researchers have used BODIPY fatty acids and phospholipids to visualize compartmentalization of specific lipid classes in Schistosoma mansoni and fungi.
- β-BODIPY FL C12-HPC (D3792) has been used to examine lipid–protein interactions involved in bacterial protein secretion via fluorescence resonance energy transfer (FRET) measurements (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).
- β-BODIPY FL C5-HPC (D3803) has been used to characterize lipid domains by fluorescence correlation spectroscopy (Fluorescence Correlation Spectroscopy (FCS)—Note 1.3), confocal laser-scanning microscopy () and near-field scanning optical microscopy.
- bis-BODIPY FL C11-PC (B7701) has BODIPY FL dye–labeled sn-1 and sn-2 acyl groups (), resulting in partially quenched fluorescence that increases when one of the acyl groups is hydrolyzed by phospholipase A1 or A2. The hydrolysis products are BODIPY FL undecanoic acid (D3862) and BODIPY FL dye–labeled lysophosphatidylcholine (Figure 13.2.9). The probe has been used successfully in human neutrophils, plants and zebrafish to detect phospholipase A activity (Probes for Lipid Metabolism and Signaling—Section 17.4).
- β-BODIPY FL C5-HPC (D3803) has been used to investigate the cellular uptake of antineoplastic ether lipids.
Figure 13.2.9 Mechanism of phospholipase activity–linked fluorescence enhancement responses of bis-BODIPY FL C11-PC (B7701) and PED6 (D23739). Note that enzymatic cleavage of bis-BODIPY FL C11-PC yields two fluorescent products, whereas cleavage of PED6 yields only one.
The fluorescent phospholipid analog β-DPH HPC (D476) comprises diphenylhexatriene propionic acid coupled to glycerophosphocholine at the sn-2 position (). It is therefore related to the neutral membrane probe DPH and the cationic derivative TMA-DPH (D202, T204; Other Nonpolar and Amphiphilic Probes—Section 13.5). DPH and its derivatives exhibit strong fluorescence enhancement when incorporated into membranes, as well as sensitive fluorescence polarization (anisotropy) responses to lipid ordering (Fluorescence Polarization (FP)—Note 1.4). β-DPH HPC was originally devised to improve the localization of DPH in membranes. Unlike underivatized DPH, it can be used to specifically label one leaflet of a lipid bilayer, facilitating analysis of membrane asymmetry.
DPH derivatives are predominately used to investigate the structure and dynamics of the membrane interior either by fluorescence polarization or lifetime measurements. Researchers have used β-DPH HPC as a probe for lipid–protein interactions, alcohol-induced perturbations of membrane structure, molecular organization and dynamics of lipid bilayers and lipid peroxidation. Fluorescence lifetime measurements of β-DPH HPC provide a sensitive indicator of membrane fusion. In addition to membrane fusion, β-DPH HPC has been used to monitor various other lipid-transfer processes.
Our acyl-modified nitrobenzoxadiazole (NBD) phospholipid probes include both the NBD hexanoyl- and NBD dodecanoyl-glycerophosphocholines (NBD C6-HPC, N3786; and NBD C12-HPC, N3787). Spectral properties of some lipid probes—Table 13.2 compares the spectral properties of these probes with those of the BODIPY, DPH and pyrene lipid probes. Unlike the BODIPY phospholipids, the location of the relatively polar NBD fluorophore of NBD C6-HPC and NBD C12-HPC in phospholipid bilayers does not appear to conform to expectations based on the probe structure. A variety of physical evidence indicates that the NBD moiety "loops back" to the head-group region (Figure 13.2.1B). In fact, the fluorophore in this acyl-modified phospholipid appears to probe the same location as does the head group–labeled glycerophosphoethanolamine derivative NBD-PE (N360, see below).
These NBD probes transfer spontaneously between membranes, with NBD C6-HPC transferring more rapidly than its more lipophilic C12 analog. NBD C6-HPC can be readily removed (back-exchanged) from the plasma membrane by incubating the labeled cells either with unlabeled lipid vesicles or with bovine serum albumin. This property is useful for quantitating lipid transfer and for studying phospholipid distribution asymmetry and transmembrane "flip-flop" rates in lipid bilayers.
NBD acyl–modified probes are used for investigating lipid traffic, either by directly visualizing NBD fluorescence, by exploiting NBD self-quenching or by fluorescence resonance energy transfer methods. Lateral domains in model monolayers, bilayers and cell membranes have been characterized using NBD phospholipids in conjunction with fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) and direct microscopy techniques. Transmembrane lipid distribution (Lipid-Mixing Assays of Membrane Fusion—Note 13.1) has been assessed using fluorescence resonance energy transfer from NBD HPC to rhodamine DHPE (L1392) or alternatively by selective dithionite (S2O42–) reduction of NBD phospholipids in the outer membrane monolayer (Figure 13.2.10).
Figure 13.2.10 Dithionite reduction of 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X, N316). The elimination of fluorescence associated with this reaction, coupled with the fact that extraneously added dithionite is not membrane permeant, can be used to determine whether the NBD fluorophore is located in the external or internal monolayer of lipid bilayer membranes.
Phospholipid analogs with pyrene-labeled sn-2 acyl chains () are among the most popular fluorescent membrane probes. We offer pyrenedecanoyl-labeled glycerophospholipids with phosphocholine (H361) and phosphoglycerol (H3809) head groups.
The spectral properties of the pyrene lipid probes are summarized in Spectral properties of some lipid probes—Table 13.2. Of primary importance in terms of practical applications is the concentration-dependent formation of excited-state pyrene dimers (excimers), which exhibit a distinctive red-shifted emission (peak ~470 nm) (Figure 13.2.3).
The excimer-forming properties of pyrene are well suited for monitoring membrane fusion (Lipid-Mixing Assays of Membrane Fusion—Note 13.1) and phospholipid transfer processes. The monomer/excimer emission ratio can also be used to characterize membrane structural domains and their dependence on temperature, lipid composition and other external factors. Pyrenedecanoyl glycerophosphocholine (β-py-C10-HPC, H361) has been used to elucidate the effect of extrinsic species such as Ca2+, platelet-activating factor, drugs, membrane-associated proteins and ethanol on lipid bilayer structure and dynamics. The anionic phosphoglycerol analog (H3809, ) is preferred as a substrate for secretory phosholipases A2 relative to other phospholipid classes. The long excited-state lifetime of pyrene (Spectral properties of some lipid probes—Table 13.2) renders the fluorescence of its conjugates very susceptible to oxygen quenching, and consequently these probes can be used to measure oxygen concentrations in solutions, lipid bilayers and cells.
Glycerophospholipids in which both alcohols are esterified to pyrene fatty acids (Figure 13.2.1C), as in our bis-(1-pyrenebutanoyl)- and bis-(1-pyrenedecanoyl)glycerophosphocholines (B3781, ; B3782) show strong excimer fluorescence, with maximum emission near 470 nm. Hydrolysis of either fatty acid ester by a phospholipase results in liberation of a pyrene fatty acid and an emission shift to shorter wavelengths, making these probes useful as phospholipase substrates (Probes for Lipid Metabolism and Signaling—Section 17.4).
Phospholipid with a Dansyl-Labeled Head Group
The phospholipid analog incorporating the environment-sensitive dansyl fluorophore (dansyl DHPE, D57; ) is a useful probe of lipid–water interfaces. It is sensitive to the interactions of a number of proteins, including protein kinase C, annexins and phospholipase A2, with membrane surfaces. Dansyl DHPE has also been used to examine the effects of cholesterol on the accessibility of the dansyl hapten to antibodies (Antibodies for Detecting Membrane-Surface Labels—Note 13.2).
Phospholipid with a Marina Blue Dye–Labeled Head Group
Marina Blue DHPE (M12652, ) is optimally excited by the intense 365 nm spectral line of the mercury-arc lamp and exhibits bright blue fluorescence emission near 460 nm. Significantly, the pKa value of this 6,8-difluoro-7-hydroxycoumarin derivative is 2–3 log units lower than that of nonfluorinated 7-hydroxycoumarin analogs; consequently, Marina Blue DHPE is expected to be strongly fluorescent in membranes, even at neutral pH.
Phospholipid with a Pacific Blue Dye–Labeled Head Group
The Pacific Blue dye–labeled phospholipid (Pacific Blue DMPE, P22652; ) is our only head group–labeled phospholipid with tetradecanoyl (myristoyl) esters rather than hexadecanoyl (palmitoyl) esters. This blue-fluorescent phospholipid is structurally similar to a phospholipid described by Gonzalez and Tsien for use in a FRET-based measurement of membrane potential.
Phospholipid with an NBD-Labeled Head Group
The widely used membrane probe nitrobenzoxadiazolyldihexadecanoylglycerophosphoethanolamine (NBD-PE, N360; ) has three important optical properties: photolability, which makes it suitable for photobleaching recovery measurements; concentration-dependent self-quenching; and fluorescence resonance energy transfer to rhodamine acceptors (usually rhodamine DHPE, L1392). Spectroscopic characteristics of NBD-PE are generally similar to those described for our phospholipids with NBD-labeled acyl chains (N3786, N3787). NBD-PE is frequently used in NBD–rhodamine fluorescence energy transfer experiments to monitor membrane fusion (Lipid-Mixing Assays of Membrane Fusion—Note 13.1). In addition, this method can be used to detect lipid domain formation and intermembrane lipid transfer and to determine the transbilayer distribution of phospholipids. Attachment of the NBD fluorophore to the head group makes NBD-PE resistant to transfer between vesicles. NBD-PE has been used in combination with either rhodamine DHPE (L1392) or Texas Red DHPE (T1395MP) for visualizing the spatial relationships of lipid populations by fluorescence resonance energy transfer microscopy. The nitro group of NBD can be reduced with sodium dithionite, irreversibly eliminating the dye's fluorescence (Figure 13.2.10). This technique can be employed to determine whether the probe is localized on the outer or inner leaflet of the cell membrane. The argon-ion laser–excitable NBD-PE is also a frequent choice for fluorescence recovery after photobleaching (FRAP) measurements of lateral diffusion in membranes. In addition, NBD-PE is of particular value for monitoring bilayer-to-hexagonal phase transitions, because these transitions cause an increase in NBD-PE's fluorescence intensity.
Phospholipid with a Fluorescein-Labeled Head Group
Fluorescein-derivatized dihexadecanoylglycerophosphoethanolamine (fluorescein DHPE, F362; ) is a membrane-surface probe that is sensitive to both the local electrostatic potential and pH. An anti–fluorescein/Oregon Green dye antibody (A889, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) has been employed in combination with fluorescein DHPE to investigate specific recognition interactions at membrane surfaces (Antibodies for Detecting Membrane-Surface Labels—Note 13.2).
Because of fluorescein's photolability, fluorescein DHPE is a useful reagent for measuring lateral diffusion in membranes using fluorescence photobleaching recovery methods. Another technique, single-particle tracking (SPT), provides direct measurements of diffusion rates by calculating the trajectories of fluorescent polystyrene beads or colloidal gold particles from time-sequential images. FluoSpheres fluorescent microspheres (Microspheres—Section 6.5) were labeled with streptavidin and then coupled to fluorescein DHPE using a biotinylated conjugate of anti–fluorescein/Oregon Green monoclonal 4-4-20 (A6421, Anti-Dye and Anti-Hapten Antibodies—Section 7.4). Diffusion rates measured with this bridged conjugate in glass-supported phospholipid bilayers were the same as those determined with streptavidin beads coupled directly to biotin-X DHPE (B1616). Fluorescein DHPE has also been used in conjunction with polyclonal anti–fluorescein/Oregon Green dye antibody (A889, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) to prepare colloidal gold probes for SPT diffusion measurements in supported phospholipid bilayers and in keratocyte plasma membranes.
Phospholipid with an Oregon Green 488 Dye–Labeled Head Group
With absorption and emission spectra that are virtually superimposable on those of fluorescein, our Oregon Green 488 DHPE (O12650, ) provides an important alternative to fluorescein DHPE in its many applications. When compared with the fluorescein derivative, Oregon Green 488 DHPE exhibits greater photostability and a lower pKa (pKa = 4.7 versus 6.4 for fluorescein); however, these pKa values may differ when the probes are bound to membranes.
Phospholipid with a BODIPY FL Dye–Labeled Head Group
Our phospholipid with the green-fluorescent BODIPY FL dye attached to the head group (BODIPY FL DHPE, D3800; ) has significant potential for studies of molecular recognition interactions at membrane surfaces (Antibodies for Detecting Membrane-Surface Labels—Note 13.2). Spectral properties of this BODIPY probe is generally the same as those described above for phospholipids with BODIPY FL dye–labeled acyl chains.
Phospholipids with a Rhodamine or Texas Red Dye–Labeled Head Group
The rhodamine-labeled phospholipids TRITC DHPE (T1391, ) and rhodamine DHPE (often referred to as N-Rh-PE, L1392; Figure 13.2.1G) do not readily transfer between separated lipid bilayers. This property has led to the extensive use of rhodamine DHPE for membrane fusion assays based on fluorescence resonance energy transfer from NBD-PE (Lipid-Mixing Assays of Membrane Fusion—Note 13.1). In addition, these probes are good resonance energy transfer acceptors from fluorescent lipid analogs such as the BODIPY and NBD phospholipids and from protein labels such as 5-iodoacetamidofluorescein 5-IAF, I30451; Thiol-Reactive Probes Excited with Visible Light—Section 2.2) and IAEDANS (I14, Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3). Rhodamine-labeled phospholipids have also been used as tracers for membrane traffic during endocytosis and for lipid processing in hepatocytes. Texas Red DHPE (T1395MP) is principally employed as an energy transfer acceptor from NBD, BODIPY and fluorescein lipid probes. The longer emission wavelength of the Texas Red dye provides superior separation of the donor and acceptor emission signals in resonance energy transfer microscopy. This technique has enabled visualization of ATP-dependent fusion of liposomes with the Golgi apparatus. Membrane flux during hemagglutinin-mediated cell–cell fusion has been visualized using Texas Red DHPE and the lipophilic carbocyanine DiI (D282, D3911; Dialkylcarbocyanine and Dialkylaminostyryl Probes—Section 13.4) as membrane labels.
Phospholipids with a Biotinylated Head Group
We offer phospholipids labeled with a biotin at the head group to facilitate binding of labeled membranes to other biomolecules. The biotinylated phospholipids (biotin DHPE, B1550; biotin-X DHPE, B1616, ) can be used to couple avidin or streptavidin (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.10) to cell membranes, liposomes and lipid monolayers. Avidin can then be employed as a bridge for antibody coupling or for assembling liposomes into multiplex structures. Liposomes incorporating biotinylated phospholipids can also be used to immobilize membrane-bound proteins for analysis by affinity chromatography. Interactions of biotinylated lipids with streptavidin provide a model for molecular recognition processes at membrane surfaces. The phase structure of lipid assemblies incorporating biotinylated phospholipids has been studied by X-ray diffraction, 31P NMR and differential scanning calorimetry.
With the resolution inherent in an image-based methodology and the productivity of high-throughput assays, high-content screening (HCS) or automated imaging provides a powerful tool for studying biology in a spatial and temporal context. Using HCS technology, researchers can examine multiple cellular targets and parameters in a large number of individually imaged cells and quantitatively assess the data. While many Molecular Probes products can be directly applied to HCS protocols, we have developed validated tools and assays specifically for HCS platforms. These HCS products are:
- Validated on multiple imaging platforms
- Packaged in automation-compatible formulations
- Compatible with multiplex applications
Although designed for HCS platforms, HCS products and kits can also be used with conventional fluorescence microscopes equipped with standard optical filter sets.
HCS LipidTOX Phospholipidosis Detection Reagents
Phospholipidosis is often triggered by cationic amphiphilic drugs, which become enriched in lysosomes to high concentrations and inhibit normal metabolism of phospholipids. The subsequent intracellular accumulation of phospholipids and formation of lamellar bodies—phospholipidosis—can be detected in cells incubated in the presence of phospholipids conjugated to fluorescent dyes.
HCS LipidTOX Green and HCS LipidTOX Red phospholipidosis detection reagents (H34350, H34351), also called LipidTOX phospholipid stains, were specifically developed to characterize the potentially toxic side effects of compounds on lipid metabolism in mammalian cell lines using image-based HCS assays. Key advantages of this series of phospholipidosis detection reagents over conventional stains such as NBD-PE (N360) include their ready-to-use aqueous formulation, their narrow emission profiles (excitation/emission maxima ~495/525 nm for HCS LipidTOX Green phospholipidosis detection reagent and ~595/615 nm for HCS LipidTOX Red phospholipidosis detection reagent) and their compatibility with HCS LipidTOX neutral lipid stains (see below).
HCS LipidTOX phospholipidosis detection reagents have not been observed to affect the normal growth of cells, and their live-cell staining patterns are maintained after formaldehyde fixation. These reagents are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. HCS LipidTOX phospholipidosis detection reagents can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets.
HCS LipidTOX Neutral Lipid Stains
As with phospholipidosis, steatosis or the intracellular accumulation of neutral lipids as lipid droplets or globules is often triggered by drugs that affect the metabolism of fatty acids or neutral lipids. HCS LipidTOX neutral lipid stains were developed to characterize the effects of drugs and other compounds on lipid metabolism in mammalian cell lines. HCS LipidTOX neutral lipid stains have an extremely high affinity for neutral lipid droplets. These reagents are added after cell fixation and do not require subsequent wash steps after incubation with the sample. Key advantages of this series of neutral lipid stains over conventional stains such as nile red (N1142, Other Nonpolar and Amphiphilic Probes—Section 13.5) include their ready-to-use formulations, their flexibility for multiplexing protocols and their compatibility with HCS LipidTOX phospholipidosis detection reagents (see above). HCS LipidTOX neutral lipid stains can also be used to monitor the formation and differentiation of adipocytes, a process called adipogenesis. Adipogenesis is of acute interest to the biomedical and drug discovery community as it plays an important role in diseases such as obesity, diabetes and atherosclerosis.
Described more thoroughly in Other Nonpolar and Amphiphilic Probes—Section 13.5, HCS LipidTOX neutral lipid stains are available with green, red and deep red fluorescence emission:
HCS LipidTOX neutral lipid stains are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. These stains can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets.
Figure 13.2.11 LipidTOX Green neutral lipid stain and fatty acid–binding protein (FABP4) antibody labeling in adipocytes. Adipocytes differentiated from 3T3-L1 mouse fibroblasts were fixed with formaldehyde and permeabilized with saponin before labeling with rabbit anti–fatty acid binding protein (FABP4) IgG (red). These cells were then stained with LipidTOX Green neutral lipid stain (H34475, green), counterstained with DAPI (D1306, D21490; blue) and mounted in ProLong Gold antifade reagent (P36930).
HCS LipidTOX Phospholipidosis and Steatosis Detection Kit
The detection and analysis of pre-lethal mechanisms in toxicological profiling and compound screening are extremely important components of the drug discovery process. The cationic amphiphilic drugs are among the most prominent examples of compounds that impact lipid metabolism of cells. These drugs tend to become enriched in lysosomes to high concentrations and inhibit the normal metabolism of phospholipids, which in turn causes the intracellular accumulation of phospholipids and the formation of lamellar bodies. Other drug classes more adversely affect various aspects of fatty acid or neutral lipid metabolism, leading to the cytoplasmic accumulation of neutral lipid as lipid droplets or globules.
The HCS LipidTOX Phospholipidosis and Steatosis Detection Kit (H34157, H34158) provides a complete set of reagents for performing validated HCS assays to detect and distinguish these two facets of cytotoxicity—the intracellular accumulation of phospholipids (phospholipidosis) and of neutral lipids (steatosis)—in mammalian cell lines after exposure to test compounds. This kit includes an aqueous, red-fluorescent formulation of labeled phospholipids (LipidTOX Red phospholipid stain, excitation/emission ~595/615 nm) and a ready-to-use, highly selective green-fluorescent stain for neutral lipids (LipidTOX Green neutral lipid stain, excitation/emission ~495/505 nm), which can be used sequentially for the analysis of phospholipidosis and steatosis, respectively, or can be used separately for single-parameter analysis.
After incubation with LipidTOX Red phospholipid stain and a test compound, the cells are fixed with formaldehyde and labeled with LipidTOX Green neutral lipid stain (Figure 13.2.12). Neither LipidTOX Red phospholipid stain, nor LipidTOX Green phospholipid stain described above, requires sonication or organic solvents. Furthermore, LipidTOX Green neutral lipid stain (as well as the other LipidTOX neutral lipid stains described above) is more selective than nile red, allowing you to easily distinguish neutral lipids (such as those in adipocytes and cells undergoing steatosis) from other types of lipids.
Each HCS LipidTOX Phospholipidosis and Steatosis Detection Kit provides:
- LipidTOX Red phospholipid stain
- LipidTOX Green neutral lipid stain
- Hoechst 33342 for nuclear labeling
- Propranolol, a positive-control compound for inducing phospholipidosis
- Cyclosporin A, a positive-control compound for inducing steatosis
- Dimethylsulfoxide (DMSO)
- Detailed protocols (HCS LipidTOX Phospholipidosis and Steatosis Detection Kit)
Sufficient reagents are provided for 240 assays (H34157, 2-plate size) or 1200 assays (H34158, 10-plate size), based on assay volumes of 100 µL per well. These kits are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. The fluorescent stains used for the analysis of phospholipidosis and steatosis can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets.
Figure 13.2.12 Multiplex detection of phospholipidosis and steatosis in HepG2 cells using the HCS LipidTOX Phospholipidosis and Steatosis Detection Kit (H34157, H34158). HepG2 cells were co-incubated with tamoxifen and LipidTOX Red phospholipid stain, followed by fixation with formaldehyde and labeling with HCS LipidTOX Green neutral lipid stain and Hoechst 33342 (H1399, H3570, H21492).
|B7701||1029.80||FF,D,L||see Notes||505||123,000||512||MeOH||2, 6|
|D476||782.01||FF,D,L||see Notes||354||81,000||428||MeOH||2, 7|
|D3792||895.95||FF,D,L||see Notes||506||86,000||513||EtOH||2, 5|
|D3793||881.93||FF,D,L||see Notes||509||86,000||513||EtOH||2, 5|
|D3800||1067.23||FF,D,L||see Notes||505||87,000||511||MeOH||2, 5|
|D3803||797.77||FF,D,L||see Notes||503||80,000||512||MeOH||2, 5|
|D3805||746.68||FF,D,L||see Notes||504||79,000||511||MeOH||2, 5|
|D3815||921.91||FF,D,L||see Notes||534||64,000||552||MeOH||2, 5|
|F362||1182.54||FF,D,L||see Notes||496||88,000||519||MeOH||2, 10|
|H361||850.13||FF,D,L||see Notes||342||37,000||376||MeOH||2, 11, 12|
|H3809||856.09||FF,D,L||see Notes||341||38,000||376||MeOH||2, 11, 12|
|M12652||944.14||FF,D,L||see Notes||365||18,000||460||MeOH||2, 10|
|N360||956.25||FF,D,L||see Notes||463||21,000||536||MeOH||2, 13|
|N3786||771.89||FF,D,L||see Notes||465||21,000||533||EtOH||2, 13|
|N3787||856.05||FF,D,L||see Notes||465||22,000||534||EtOH||2, 13|
|O12650||1086.25||FF,D,L||see Notes||501||85,000||526||MeOH||2, 10|
|P31||372.51||L||DMF, DMSO||341||43,000||377||MeOH||11, 12|
|P96||400.56||L||DMF, DMSO||341||44,000||377||MeOH||11, 12|
|P243||456.67||L||DMF, DMSO||341||43,000||377||MeOH||11, 12|
|P1903MP||288.35||L||DMF, DMSO||341||43,000||376||MeOH||11, 12|