Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures.

Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR, DNA DAMAGE CHECKPOINT, AND APOPTOSIS.

DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

Isotope-coded carbamidomethylation for quantification of N-glycoproteins with online microbore hollow fiber enzyme reactor-nanoflow liquid chromatography-tandem mass spectrometry.

This paper introduces a simple, inexpensive, and robust quantitative proteomic method for quantifying N-linked glycoproteins based on isotope-coded carbamidomethylation (iCCM) incorporated into an online microbore hollow fiber enzyme reactor and nanoflow liquid chromatography-tandem mass spectrometry (mHFER-nLC-MS/MS). The iCCM quantitation uses carbamidomethylation (CM; a routine protection of thiol groups before proteolysis) of the Cys residue of proteins with iodoacetamide (IAA) or its isotope (IAA-(13)C2,D2: 4 Da difference). CM-/iCCM-labeled proteome samples are mixed for proteolysis; then, online enrichment of N-glycopeptides using lectin affinity is carried out in an mHFER before nLC-MS/MS for quantification using multiple reaction monitoring (MRM). Initial evaluation of the iCCM method varying the mixing ratio of CM-/iCCM-labeled bovine serum albumin (BSA) standards yielded successful quantification of 18 peptides with less than 2% variation in the calculated ratio of light/heavy-labeled peptides. The iCCM quantitation with mHFER-nLC-MS/MS was evaluated with three standard glycoproteins (α-1-acid glycoproteins, fetuin and transferrin) and then applied to serum glycoproteins from liver cancer patients and controls, resulting in successful quantification of 73 N-glycopeptides (from 49 N-glycoproteins), among which 19 N-glycopeptides from 14 N-glycoproteins showed more than a 2.5-fold aberrant change in liver cancer patients' sera compared with the pooled control. Although iCCM quantitation with mHFER-nLC-MS/MS applies only to glycopeptides with Cys residue, the method can offer several advantages over other labeling methods when applied to targeted glycoproteins: The iCCM method does not require an additional labeling reaction under special conditions nor complicated procedures to purify labeled products using additional columns. Isotope labeling at the protein level can minimize potential uncertainty originating from unequal efficiencies in protein digestion in separate vials and retrieval of each labeled peptide when labeling takes place at the peptide level. In addition, the labeling reagents for the iCCM method are readily obtained at a reasonable cost, which can make protein quantification easily accessible.

Determination of phosphorus impurity that directly affects quantification of microbial genomic DNA using inductively coupled plasma optical emission spectrometry.

We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP-OES). Application of ICP-OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB). Further investigations using reversed-phase high-performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), and (31)P nuclear magnetic resonance (NMR) identified the phosphorus impurity as lipoteichoic acid (LTA).

Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography-tandem mass spectrometry.

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 µmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

Development of an online microbore hollow fiber enzyme reactor coupled with nanoflow liquid chromatography-tandem mass spectrometry for global proteomics.

In this study, we report the development of a microbore hollow fiber enzyme reactor (mHFER) coupled to nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) for the online digestion or selective enrichment of glycopeptides and analysis of proteins. With mHFER, enzymatic digestion of protein could be achieved by continuous flow within a very small volume (~10 µL) of mHF inserted in a PEEK tube. Digested peptides exited through the pores of the hollow fiber membrane wall to external single or multiplexed trap columns for nLC-ESI-MS/MS analysis. Evaluation of online mHFER-nLC-ESI-MS/MS system was made with bovine serum albumin (BSA) by varying the temperature of digestion and the amount of protein injected. We evaluated the ability of the mHFER system to enrich glycopeptides by injecting a mixture of lectin (concanavalin A) and digested peptides from α-1-acid glycoprotein (AGP) into the mHFER, followed by delivery of PNGase F for endoglycosidic digestion. Nonglycosylated peptides unbound to lectins eluted at the first breakthrough run while N-linked glycopeptides eluted after the endoglycosidic digestion. The developed method was applied to urine samples from patients with prostate cancer and controls; 67 N-linked glycopeptides were identified and relative differences in glycopeptide content between patient and control samples were determined.

Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.

The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards.

Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface.

We describe a capillary electrophoresis-mass spectrometry (CE-MS) method for newborn screening of a representative amino acid metabolic disease, namely, phenylketonuria (PKU). Underivatized phenylalanine and tyrosine in a dried blood spot (DBS) were simultaneously determined by CE-MS equipped with an ionophore membrane-packed sheathless electrospray ionization interface, which was developed by our group. The method was optimized for rapid determination of the underivatized amino acids, phenylalanine and tyrosine extracted from a DBS. Under the optimized conditions, the limit of detection of phenylalanine and tyrosine (signal-to-noise ratio, 3) was 0.03 and 0.07 mg/L in DBS, respectively, with a CE run time of less than 3 min. For repeated runs of a sample, coefficients of variation (CVs) for migration time were less than 3.7%, whereas CVs for the area ratio under the curve were 2.1 and 2.9% for 20 consecutive runs of 49.5 mg/kg Phe and 36.2 mg/kg Tyr, respectively. However, the relative standard deviations of intra- and interday assays for DBS samples were <6.2 and <5.8%, respectively, which were substantially due to sample extraction from DBS. The analytical method was applied to real clinical samples of Korean neonates, and results were compared with those of conventional methods for PKU diagnosis, which required reference analytical methods such as isotope dilution CE-MS or high-performance liquid chromatography-mass spectrometry for quality assurance of the conventional kit-based assays. The distinct advantages of high sensitivity and extremely low sample volume, as well as a simple, easy, and economic sample pretreatment, were demonstrated for the proposed method.

Identification of potential serum biomarkers in mercury-treated mice using a glycoproteomic approach.

Mercury is a well-recognized health hazard and a deleterious environmental contaminant. Exposure to mercury can cause neurotoxic manifestations, nephrotoxicity, and immune function alterations; however, the mechanisms and related proteins responsible for these effects are still unclear. Our goal is to understand the relationship between the toxicity of mercury and the proteins affected by this toxic heavy metal and to define biomarkers for mercury intoxication. Two different forms of mercury, organic methylmercury or inorganic mercury sulfide, were orally administered to the mice for 4 weeks. To reduce complexity of the serum proteome, we enriched glycoproteins from mice serum with lectin concanavalin A resin, and the tryptic peptides of the purified glycoproteins were subjected to nanoultra performance liquid chromatography-Quadrupole time-of-flight for identification and label-free quantification. In this study, we characterized approximately 209 proteins from mice serum, and, among them, 21 proteins were differentially expressed in organic methylmercury-treated mice serum compared with the control group. Two proteins, serum amyloid P component (SAP) and inter α-trypsin inhibitor heavy chain 4 (ITI-H4), were upregulated in organic methylmercury-treated mice and confirmed with different doses of both types of mercury by Western blot analysis. Results of immunohistochemistry also confirmed the validity of SAP and ITI-H4 as biomarker candidates for organic methylmercury exposure. Findings of this study may assist in understanding the relationship between toxicity of mercury and upregulated proteins in mouse serum. Furthermore, the proteins identified here might be used as biomarker candidates in mercury intoxication.

Dual lectin-based size sorting strategy to enrich targeted N-glycopeptides by asymmetrical flow field-flow fractionation: profiling lung cancer biomarkers.

A dual lectin-based size sorting and simultaneous enrichment strategy for selectively isolating N-linked glycopeptides was developed using asymmetrical flow field-flow fractionation (AF4). AF4 is an elution-based method for separating biological macromolecules that has been utilized for the separation of lectin-glycopeptide complexes formed by mixing serum peptides with lectin cocktails according to the difference in diffusion coefficients. It has also been used for simultaneous depletion of nonglycosylated peptides. The dual lectin-based enrichment method was applied to proteolytic peptides from lung cancer serum samples with two lectins (WGA, GlcNAc-specific, and SNA, Sia-specific), and the whole mixture was separated by AF4. The lectin-glycopeptide complex fractions collected during AF4 separation were endoglycosidically digested with PNGase F. The resulting deamidated glycopeptides were analyzed by nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) to semiquantitatively profile the N-linked glycopeptides from the sera of lung cancer patients and healthy controls. The AF4 enrichment strategy coupled with nLC-ESI-MS-MS identified 16/24 (up/down-regulated by at least 10-fold compared to normal sera) N-linked glycopeptides from a WGA complex fraction of lung cancer sera and 18/3 from a SNA fraction.

Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids.

A high durability sheathless electrospray ionization interface of CE-MS is applied for the sensitive analysis of underivatized amino acids. The sheathless interface was realized using an ionophore membrane-packed electro-conduction channel. The interface functioned well with a volatile alkaline background electrolyte (BGE) and uncoated fused-silica capillaries for CE-MS analysis of underivatized amino acids. High electroosmotic flow with alkaline BGE facilitated high separation efficiency (>100,000 theoretical plates) and short analysis time (<15 min). Both the short-term stability and long-term durability are particularly suited for routine applications. Using electrokinetic injection and the multiple reaction monitoring (MRM) mode with a triple-quadrupole analyzer, high sensitivity was achieved, which yielded detection limits of 0.05-0.81 µM. For the quantitation of underivatized amino acids, quantification precisions (RSDs) for intra- and inter-day analyses were less than 3%. Recoveries from serum were 96.3-101.8% for isotope dilution mass spectrometry (IDMS). When compared with HPLC-IDMS for human serum samples, highly agreeable (96.9-102.0%) results were obtained with the proposed CE-IDMS method.

Induction-driven stabilization of the anion-π interaction in electron-rich aromatics as the key to fluoride inclusion in imidazolium-cage receptors.

Intermolecular interactions that involve aromatic rings are key processes in both chemical and biological recognition. It is common knowledge that the existence of anion-π interactions between anions and electron-deficient (π-acidic) aromatics indicates that electron-rich (π-basic) aromatics are expected to be repulsive to anions due to their electron-donating character. Here we report the first concrete theoretical and experimental evidence of the anion-π interaction between electron-rich alkylbenzene rings and a fluoride ion in CH(3)CN. The cyclophane cavity bridged with three naphthoimidazolium groups selectively complexes a fluoride ion by means of a combination of anion-π interactions and (C-H)(+)···F(-)-type ionic hydrogen bonds. (1)H NMR, (19)F NMR, and fluorescence spectra of 1 and 2 with fluoride ions are examined to show that only 2 can host a fluoride ion in the cavity between two alkylbenzene rings to form a sandwich complex. In addition, the cage compounds can serve as highly selective and ratiometric fluorescent sensors for a fluoride ion. With the addition of 1 equiv of F(-), a strongly increased fluorescence emission centered at 385 nm appears at the expense of the fluorescence emission of 2 centered at 474 nm. Finally, isothermal titration calorimetry (ITC) experiments were performed to obtain the binding constants of the compounds 1 and 2 with F(-) as well as Gibbs free energy. The 2-F(-) complex is more stable than the 1-F(-) complex by 1.87 kcal mol(-1), which is attributable to the stronger anion-π interaction between F(-) and triethylbenzene.

Fluorescent sensing of triphosphate nucleotides via anthracene derivatives.

A nucleotide is composed of a nucleobase, a five-carbon sugar, and phosphate groups. Recognition of these three sites can provide useful information for the development of selective fluorescent receptors for a specific nucleotide. In this paper, anthracene derivatives with two imidazolium groups at the 1,8- and 9,10-positions, quaternary ammonium groups, or the boronic acid group were examined for the recognition of nucleotides, such as ATP, GTP, CTP, TTP, UTP, ADP, and AMP, via fluorescence changes. The anthracene group provides the interaction between the bases of the nucleotides. The imidazolium and quaternary ammonium groups induce hydrogen bonding interactions with the phosphate groups of the nucleotides. The boronic acid group can interact with the ribose of the nucleotides.

Synthesis of 'clickable' acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation.

Alkynyl- and azido-tagged 3-oxo-C(12)-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C(12)-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative.

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive.

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2'-deoxyribonucleoside 5'-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.

Revisit to imidazolium receptors for the recognition of anions: highlighted research during 2006-2009.

In this tutorial review we discuss imidazolium receptors for anion recognition and recent contributions between 2006-2009 are reviewed according to target analytes, such as ATP and DNA, as well as structural classification, including cage type imidazoliums, imidazolium calixarenes, ferrocenyl imidazoliums, chiral systems, fluorescent or colorimetric imidazoliums, imidazolium cyclophane, nano assembled structures, bile acid-imidazolium, and tripodal-imidazolium systems.

Chemosensors for pyrophosphate.

The selective detection of the anion pyrophosphate (PPi) is a major research focus. PPi is a biologically important target because it is the product of ATP hydrolysis under cellular conditions, and because it is involved in DNA replication catalyzed by DNA polymerase, its detection is being investigated as a real-time DNA sequencing method. In addition, within the past decade, the ability to detect PPi has become important in cancer research. In general, the sensing of anions in aqueous solution requires a strong affinity for anions in water as well as the ability to convert anion recognition into a fluorescent or colorimetric signal. Among the variety of methods for detecting PPi, fluorescent chemosensors and colorimetric sensors for PPi have attracted considerable attention during the past 10 years. Compared with the recognition of metal ions, it is much more challenging to selectively recognize anions in an aqueous system due to the strong hydration effects of anions. Consequently, the design of PPi sensors requires the following: an understanding of the molecular recognition between PPi and the binding sites, the desired solubility in aqueous solutions, the communicating and signaling mechanism, and most importantly, selectivity for PPi over other anions such as AMP and ADP, and particularly phosphate and ATP. This Account classifies chemosensors for PPi according to topological and structural characteristics. Types of chemosensors investigated and reported in this study include those that contain metal ion complexes, metal complexes combined with excimers, those that function with a displacement approach, and those based on hydrogen-bonding interaction. Thus far, the utilization of a metal ion complex as a binding site for PPi has been the most successful strategy. The strong binding affinity between metal ions and PPi allows the detection of PPi in a 100% aqueous solution. We have demonstrated that carefully designed receptors can distinguish between PPi and ATP based on their different total anionic charge densities. We have also demonstrated that a PPi metal ion complex sensor has a bioanalytical application. This sensor can be used in a simple and quick, one-step, homogeneous phase detection method in order to confirm DNA amplification after polymerase chain reaction (PCR).

A sheathless CE/ESI-MS interface with an ionophore membrane-packed electro-conduction channel.

A robust and convenient sheathless CE/ESI-MS interface realized with an ionophore membrane-packed electro-conduction channel is described. Sheathless interfaces that may provide higher sensitivity for MS detection than sheath flow-supported interfaces generally show instability and short lifetimes due to their imperfection in making an electrical contact with the emitter tip. In this work, we designed a sheathless interface based on an ionophore membrane-packed electro-conduction channel. At the joining point of the CE capillary and the emitter capillary, the conduction channel was implemented toward the exterior of the interface body, where a platinum wire electrode was placed. The conduction channel transferred the electric field from the external Pt electrode to the joining point, but prevented the effluent of CE from leaking. The interface body was designed to have receptacles for standard capillary tubing with finger-tight fittings, which allowed easy replacement of capillary tubing. Stable electrospray was observed for an extended time period without any signs of bubbling or damage to the emitter tip. No significant increment of dead-volume at the interface was observed for well-aligned capillaries. Sensitive and stable CE-MS detection of the model compound of creatinine and uric acid was demonstrated.