Quantitative Cellular Assay Research Articles

A Toxicogenic Interaction between Intracellular Amyloid-β and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid β (Aβ) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aβ oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aβ-ApoE interaction suppresses these changes and concomitantly reduces Aβ toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aβ oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Development and Validation of High-Content Analysis for Screening HDAC6-Selective Inhibitors.

Throughout recent decades, histone deacetylase (HDAC) inhibitors have shown encouraging potential in cancer treatment, and several pan-HDAC inhibitors have been approved for treating malignant cancers. Numerous adverse effects of pan-HDAC inhibitors have been reported, however, during preclinical and clinical evaluations. To avoid undesirable responses, an increasing number of investigations are focusing on the development of isotype-selective HDAC inhibitors. In this study, we present an effective and quantitative cellular assay using high-content analysis (HCA) to determine compounds' inhibition of the activity of HDAC6 and Class I HDAC isoforms, by detecting the acetylation of their corresponding substrates (i.e., α-tubulin and histone H3). Several conditions that are critical for HCA assays, such as cell seeding number, fixation and permeabilization reagent, and antibody dilution, have been fully validated in this study. We used selective HDAC6 inhibitors and inhibitors targeting different HDAC isoforms to optimize and validate the capability of the HCA assay. The results indicated that the HCA assay is a robust assay for quantifying compounds' selectivity of HDAC6 and Class I HDAC isoforms in cells. Moreover, we screened a panel of compounds for HDAC6 selectivity using this HCA assay, which provided valuable information for the structure-activity relationship (SAR). In summary, our results suggest that the HCA assay is a powerful tool for screening selective HDAC6 inhibitors.

Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease

Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.

Antibody-conjugated mesoporous silica nanoparticles for brain microvessel endothelial cell targeting.

Brain microvessel endothelial cells (BMECs) are the main structural and dynamic components of the blood-brain barrier (BBB), preventing the majority of drugs from reaching the brain. Since BMECs are involved in a wide range of central nervous system diseases, the development of nanocarriers that trigger receptor-mediated uptake in these cells has been suggested as a promising approach to an increased drug delivery to the brain. Here, we report the size and the bioconjugation effects of antibody-conjugated mesoporous silica nanoparticles (MSNs) on in vitro and in vivo targeting ability to BMECs. For this, Ri7 antibody was conjugated to MSNs of two different sizes (50 nm and 160 nm in diameter) through a polyethylene glycol (PEG) linker. The particles were also functionalized with a MRI contrast agent (gadolinium chelate) and with a fluorescent label. The functionalized MSN suspensions showed good colloidal stability. The Ri7 antibody immobilized on the MSN surface maintained its high specific activity and high binding affinity, as demonstrated in vitro. Cells incubated with gadolinium-chelated Ri7-MSNs showed a significant MRI positive contrast enhancement, highlighting the potential of such nanoparticles for theranostic applications. To measure the uptake and affinity of Ri7-MSNs to brain endothelial and neuronal cells, cell uptake studies were performed and a quantitative cellular assay was developed. The results revealed that endocytosis of nanoparticles is mediated by transferrin receptors and that Ri7-MSN cellular uptake is size- and time-dependent. A highest specific uptake was found with 50 nm Ri7-MSNs. Upon intravenous injection, 50 nm Ri7-MSNs were specifically accumulated in BMECs, suggesting the strong potential of antibody-coated nanoparticles for targeting BMECs in vivo. These findings open the door to therapeutic targeting of BMECs, enabling potential therapeutic drug delivery to the brain.

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection.

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays.

Cell–cell interaction networks regulate blood stem and progenitor cell fate

Communication networks between cells and tissues are necessary for homeostasis in multicellular organisms. Intercellular (between cell) communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self-renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. We have developed a novel mathematical model of blood stem cell development incorporating cell-level kinetic parameters as functions of secreted molecule-mediated intercellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses, we show that blood stem and progenitor cell fate is regulated by cell–cell feedback, and can be controlled non-cell autonomously by dynamically perturbing intercellular signalling. We extend this concept by demonstrating that variability in the secretion rates of the intercellular regulators is sufficient to explain heterogeneity in culture outputs, and that loss of responsiveness to cell–cell feedback signalling is both necessary and sufficient to induce leukemic transformation in silico.

Influence of Hydrodynamic Conditions on Quantitative Cellular Assays in Microfluidic Systems

This study demonstrates the importance of the hydrodynamic environment in microfluidic systems in quantitative cellular assays using live cells. Commonly applied flow conditions used in microfluidics were evaluated using the quantitative intracellular Ca2+ analysis of Chinese hamster ovary (CHO) cells as a model system. Above certain thresholds of shear stress, hydrodynamically induced intracellular Ca2+ fluxes were observed which mimic the responses induced by chemical stimuli, such as the agonist uridine 5'-triphosphate tris salt (UTP). This effect is of significance given the increasing application of microfluidic devices in high-throughput cellular analysis for biophysical applications and pharmacological screening.

Sensitive, real-time monitoring of UV-induced stress in a single, live plant cell using an optical trap

An optical trap is used to monitor bio-deleterious effects induced by ultra-violet (UV) radiation in a single, live cell. When placed in a laser optical trap, Chlamydomonas reinhardtii cells undergo rotation because of the interplay of flagellar-based forces and optical forces generated by the laser light. Such rotation is sustained by the trapped cell's flagellar action, thereby unveiling a robust and quantitative cellular assay for flagellar function. UV induces flagellar damage, leading to dose-dependent attenuation of cellular rotation. At UV doses larger than ∼9Jm−2 rotational motion ceases. The ability of the cell to rotate in an optical trap is, therefore, a measure of the damage response of the cell. By monitoring cell rotation, we can quantify UV damage with high sensitivity, at threshold doses as low as 2Jm−2. Apart from the capability of UV-damage detection, our rotation assay is also sensitive to the cellular protective responses against such damage. To illustrate this additional facet, we quantify the efficacy of ascorbic acid in combating the UV-damage response of individual cells. Upon addition of this antioxidant, there is no cessation of rotations for doses as high as 25Jm−2. This represents the first single-cell sensor that robustly quantifies a complex pleiotropic cellular response.

Cell and cell-free transplantation experiments with a mouse reticulum cell sarcoma.

Quantitative cellular assays of the transplantability of 19 spontaneous reticulum cell sarcomas (RCS) of the BC3F1 mouse were carried out by injecting serial dilutions of monodispersed cells into syngeneic hosts through the intravenous, intracranial and subcutaneous routes. A clear relationship was found between the size of the inoculum and the incidence of tumour takes by all routes, but even for inocula of 5 million cells only 13 tumors grew in one or more of the transplanted recipients. The intravenous route of injection was found to be the most effective, both in terms of the absolute number of tumor takes and the number of cells necessary to produce a given level of takes. The survival of the animals injected intravenously was related to the number of cells received in that earlier deaths occurred with the most concentrated cell suspensions. Attempts to transmit the tumor by cell-free extracts injected into newborn or 1-month-old syngeneic hosts failed to substantiate the possible presence of a specific leukemogenic agent.

The immunoglobulin-like T cell receptor—IV: Quantitative cellular assay and partial characterization of a heavy chain cross-reactive with the Fd fragment of serum μ chain

Guinea pig and rabbit antisera to murine immunoglobulins (Igs) were assessed quantitatively for their capacity to bind to T cells and T cell products. Guinea pig antisera to normal mouse serum IgM, which had been absorbed with murine red blood cells and IgG and were shown previously to be cytotoxic for murine T cells (Burckhardt el al., 1974), bind to T and B cells as determined by indirect immunofluorescence and an indirect radioimmunoassay with 125I-labeled Staphylococcus aureus protein A. Studies involving competition radioimmunoassay using a labeled λμ myeloma protein (MOPC 104E) indicated that the guinea pig anti-μ sera react with a polypeptide determinant of the Fd-fragment, as well as with Fc-region determinants. The antisera do not react with κ or λ. light chains. Both detergent extracts of thymus cells and purified T cell Ig inhibit precipitation of the λμ protein MOPC 104E, but with different inhibition slopes, thereby suggesting a cross-reaction rather than an identity between the T cell heavy chain and μ chains. The intact T cell heavy chain has an apparent mass of about 70,000 dallons, and fragments of 50,000 and 35,000–40,000 daltons, which still bore the cross-reactive antigenic determinant, could be produced by limited proteolysis. Similar fragments could also be generated from MOPC 104E and B cell surface Igs. The 35,000–40,000 fragment lacks complex carbohydrate moieties as judged by its inability to bind to concanavalin A, and studies with MOPC 104E show that it was the Fd-like fragment of the Fab-monomer produced by tryptic proteolysis. Guinea pig anti-μ sera reacted only with lymphoid cells, including monoclonal T cells of various Ly phenotype. Quantitatively, B cells express approximately four times as much of the μ-cross-reactive determinant as do T cells. However, the lack of identity between the B and T cell products renders exact comparison difficult. The present results provide direct, quantitative evidence that murine T cells of various types express a surface component of mass approximately 70,000 daltons, which shares antigenic determinants with Fd-region fragments of serum μ chain. A lack of serological identity between this chain and B cell μ or δ, plus the presence of the molecule on monoclonal lines of T cells maintained in cell culture, provides strong evidence that this heavy chain is endogenously produced by T cells.

Differential RNA metabolism in rat brain cells fractionated by zonal centrifugation.

Combined fractionation and RNA metabolism studies were made on homoiogous cell types (neurons and glia) banded from rat brain cerebral cortex and hippocampus tissue at 40,000 rev/min for 50 minutes in a Beckman Ti 14 zonal rotor loaded with a discontinuous methyl cellulose (Methocel) and sucrose density gradient. Comparisons were made on the cellular fractions recovered from animals intracisternally injected with 15 muCi tritiated cytidine (3HCR) RNA precursor one hour before sacrifice and tissue cellular filtrate preparation. Immediate 3H pool-correction of extracted 3HRNA from the three cellular Bands recovered made possible a mathematically valid measure of 3HCR-precursor incorporation into RNA of the different cell types. Comparison of the cellular 3HRNA data by analysis of variance revealed the occurrence of differential tritium labelling of RNA in the same cell types banded from different brain regions. In particular, an important advance was demonstrated by the procedures developed in these studies for the quantitative cellular assay of brain in vivo (differential) RNA metabolism.

QUANTITATION OF THE IMMUNE REJECTION OF ALLOTRANSPLANTED AKR LEUKEMIC CELLS

AKR leukemic cells were transferred into both CD2F1 and C3H/He mice and the growth and rejection of the cells followed by a quantitative cellular assay. While the leukemic cells initially grew at the same rate in both strains of mice, the CD2F1 mice eventually rejected the cells. The time to rejection was found to be dependent upon the initial inoculum size.