Intracellular flow cytometric lipid analysis - a multiparametric system to assess distinct lipid classes in live cells.

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

A colorimetric sensor array for the classification of biologically relevant tri-, di- and mono-phosphates.

We describe here a supramolecular sensing system for the array-based classification of biologically relevant mono-, di-, and tri-phosphates in aqueous buffer. The system is based on a colorimetric indicator displacement assay (IDA) with six sensor elements. Each sensor element is comprised of an equimolar cyclic peptide-colorimetric indicator ensemble. Correct classification of the eleven analytes into mono-, di-, tri-, or pyrophosphate was achieved through both principal component analysis (PCA) and linear discriminant analysis (LDA).

Detection of cell-surface phosphatidylserine using the fluorogenic probe P-IID.

The fluorogenic probe P-IID enables the detection of cell-surface phosphatidylserine (PS) using both fluorescence imaging and flow cytometry. Here we provide a detailed protocol for the use of P-IID for the qualitative detection of externalized PS in apoptotic cells using confocal microscopy, including the real-time imaging of apoptosis upon drug treatment. We also provide a detailed method for the quantitative analysis of cell death by flow cytometry, using P-IID in conjunction with the nuclear stain propidium iodide. P-IID is superior to commonly used Annexin-V fluorophore conjugates for PS detection as it provides a "turn-on" fluorescence response, displays rapid binding kinetics and can be used at low temperature (4°C), without washing and in the absence of Ca2+ ions.

A Fluorogenic Probe for Cell Surface Phosphatidylserine Using an Intramolecular Indicator Displacement Sensing Mechanism.

The detection of externalized phosphatidylserine (PS) on the cell surface is commonly used to distinguish between living, apoptotic, and necrotic cells. The tools of choice for many researchers to study apoptosis are annexin V-fluorophore conjugates. However, the use of this 35 kDa protein is associated with several drawbacks, including temperature sensitivity, Ca2+ dependence, and slow binding kinetics. Herein, a fluorogenic probe for cell surface PS, P-IID, is described, which operates by an intramolecular indicator displacement (IID) mechanism. An intramolecularly bound coumarin indicator is released in the presence of cell surface PS, leading to a fluorescence "turn-on" response. P-IID demonstrates superior performance when compared to annexin V, for both fluorescence imaging and flow cytometry. This allows P-IID to be used in time-lapse imaging of apoptosis using confocal laser scanning microscopy and demonstrates the utility of the IID mechanism in live cells.

Deltamides and Croconamides: Expanding the Range of Dual H-bond Donors for Selective Anion Recognition.

Dual H-bond donors are widely used as recognition motifs in anion receptors. We report the synthesis of a library of dual H-bond receptors, incorporating the deltic and croconic acid derivatives, termed deltamides and croconamides, respectively, and a comparison of their anion binding affinities (for monovalent species) and Brønsted acidities to those of the well-established urea and squaramide dual H-bond donor motifs. For dual H-bonding cores with identical substituents, the trend in Brønsted acidity is croconamides>squaramides>deltamides>ureas, with the croconamides found to be 10-15 pKa units more acidic than the corresponding ureas. In contrast to the trends displayed by ureas, deltamides and squaramides, N,N'-dialkyl croconamides displayed higher binding affinity to chloride than the N,N'-diaryl derivatives, which was attributed to partial deprotonation of the N,N'-diaryl derivatives at neutral pH. A number of differences in anion binding selectivity were observed upon comparison of the dual H-bond cores. Whereas the squaramides display similar affinity for both chloride and acetate ions, the ureas have significantly higher affinity for acetate than chloride ions and the deltamides display higher affinity for dihydrogenphosphate ions than other oxoanions or halides. These inherent differences in binding affinity could be exploited in the design of anion receptors with improved ability to discriminate between monovalent anions.

Quantum Chemical Prediction of Equilibrium Acidities of Ureas, Deltamides, Squaramides, and Croconamides.

Robust quantum chemical methods are employed to predict the pKa's of several families of dual hydrogen-bonding organocatalysts/anion receptors, including deltamides and croconamides as well as their thio derivatives. The average accuracy of these predictions is ∼1 pKa unit and allows for a comparison of the acidity between classes of receptors and for quantitative studies of substituent effects. These computational insights further explain the relationship between pKa and chloride anion affinity of these receptors that will be important for designing future anion receptors and organocatalysts.

Fluorescent sensing arrays for cations and anions.

Array-based sensing methods can be used to distinguish sets of similar analytes, by using a number of non-specific or cross-reactive probes. Following subsequent statistical analysis, patterns or components can be isolated that can be used to unambiguously identify the specific analyte(s) present. Over the past two decades, various arrays for the identification of cations and anions have been reported. These often employ fluorescence methods, owing to good sensitivity and a versatile, easy to read output. The past few years have seen an increase in the number of such studies reported in the literature. This critical review will summarise this recent work, and identify the criteria required for a successful array system. In particular, it will focus on the different types of molecular structures that can be used, the breadth of ions that can be distinguished in a single array, the sensitivity and dynamic range to which they can be identified, and how successfully these aims have been met.

Selective sensing of pyrophosphate in physiological media using zinc(II)dipicolylamino-functionalised peptides.

A series of linear peptide based anion receptors, in which the distance between the bis[zinc(II)dipicolylamine] binding sites and the peptide backbone was varied systematically, was prepared and their anion binding ability was investigated using indicator displacement assays. Shortening the distance between the binding site and the peptide backbone was found to enhance both the receptor affinity and selectivity for pyrophosphate over other organic polyphosphate anions in Krebs buffer with the maximum selectivity and affinity observed with a spacer length of two methylene units. The suitability of these receptors for the determination of pyrophosphate concentrations in Krebs buffer and in artificial urine was examined.