SEARCH · Search PubMed

Results for “Cell-based assay”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed1987

Teratological research using in vitro systems. IV. Cells in culture.

Several in vitro cellular systems designed to screen agents for teratogenic potential are described in this report. These assays were selected from a review of literature published through the spring of 1986 that generated over 100 references on teratological research using cell-based systems. Some of the assays have a broader application than others, but most require confirmation by one or more additional complementary tests because of the specificity of the teratogenic mechanism the assays are investigating. Included are systems that use analysis of tumor cell attachment; intercellular communication; growth of human embryonic palatal mesenchyme cells; progesterone production in porcine granulosa cells; differentiation of embryonic neural crest, limb bud, midbrain, and Drosophila cells; and differentiation of tumor cells. Because of the dynamic nature of cell culture work, the group of assays listed here should not be viewed as encompassing all cell systems of value with regard to teratogenicity testing; instead, the list represents several of the more prominent systems now being evaluated by the scientific community.

Animals↗

PubMed1993

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium.

OBJECTIVE: To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. METHODS: Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. RESULTS: Treatment of HSE with interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-gamma (IFN gamma) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFN gamma and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN gamma also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNF beta/lymphotoxin. Immunoprecipitation of TNF alpha + IFN gamma-stimulated, 125I-labeled HSE cells with anti-ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFN gamma alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. CONCLUSION: These studies indicate that IFN gamma plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

Adolescent↗

PubMed1995

Disparate cytokine regulation of ICAM-1 in rat alveolar epithelial cells and pulmonary endothelial cells in vitro.

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells in the normal lung and is induced in vitro as type II cells spread in primary culture. In contrast, in most nonhematopoetic cells ICAM-1 expression is induced in response to inflammatory cytokines. We have formed the hypothesis that the signals that control ICAM-1 expression in alveolar epithelial cells are fundamentally different from those controlling expression in most other cells. To test this hypothesis, we have investigated the influence of inflammatory cytokines on ICAM-1 expression in isolated type II cells that have spread in culture and compared this response to that of rat pulmonary artery endothelial cells (RPAEC). ICAM-1 protein, determined both by a cell-based enzyme-linked immunosorbent assay and by Western blot analysis, and mRNA were minimally expressed in unstimulated RPAEC but were significantly induced in a time- and dose-dependent manner by treatment with tumor necrosis factor-alpha, interleukin-1 beta, or interferon-gamma. In contrast, these cytokines did not influence the constitutive high level ICAM-1 protein expression in alveolar epithelial cells and only minimally affected steady-state mRNA levels. ICAM-1 mRNA half-life, measured in the presence of actinomycin D, was relatively long at 7 h in alveolar epithelial cells and 4 h in RPAEC. The striking lack of response of ICAM-1 expression by alveolar epithelial cells to inflammatory cytokines is in contrast to virtually all other epithelial cells studied to date and supports the hypothesis that ICAM-1 expression by these cells is a function of cellular differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Rat serum inhibits progression of alveolar epithelial cells toward the type I cell phenotype in vitro.

Serum contains a number of polypeptide growth factors, hormones, and soluble matrix components and may influence the state of differentiation of epithelial cells in general and of alveolar epithelial cells (AEC) in particular. To evaluate the influence of sera on the transition from the type II toward the type I cell phenotype, we compared the effects of newborn bovine serum (NBS) and rat serum (RS) on morphologic changes and expression of a type I cell-specific epitope in AEC monolayers with time in primary culture. Rat type II AEC were harvested and cultured in defined serum-free medium (MDSF), MDSF + RS (5%), or MDSF + NBS (10%). Monolayer integrity was monitored by measuring transepithelial resistance (approximately 2,000 omega.cm2) and short-circuit current (approximately 4 microA/cm2). Binding of the type I cell-specific monoclonal antibody VIIIB2 was assessed between day 1 and day 11 by cell-based enzyme-linked immunosorbent assay (ELISA) and by immunoelectron microscopy (IEM). By ELISA, in MDSF and MDSF + NBS, VIIIB2 binding increased markedly after day 2, rising approximately 4-fold by day 8 (compared with day 1). In dramatic contrast, there was essentially no increase in VIIIB2 binding through day 11 in MDSF + RS. Results from IEM for apical surface binding of VIIIB2 were similar to those obtained by ELISA. Some morphologic differences were also noted, with cells in MDSF + RS being somewhat less spread at later times than those in MDSF or MDSF + NBS. These data indicate that the rate of rat type II AEC differentiation toward the type I cell phenotype is significantly modulated by soluble factor(s) present in rat serum.

Animals↗

PubMed1994

Assays for recombinant proteins: a problem in non-linear calibration.

Quantification of protein levels in biological matrices such as serum or plasma frequently relies on the techniques of immunoassay or bioassay. The relevant statistical problem is that of non-linear calibration, where one estimates analyte concentration in an unknown sample from a calibration curve fit to known standard concentrations. This paper discusses a general framework for calibration curve fit to known standard concentrations. This paper discusses a general framework for calibration inference, that of the non-linear mixed effects model. Within this framework, we consider two issues in depth: accurate characterization of intra-assay variation, and the use of empirical Bayes methods in calibration. We show that proper characterization of intra-assay variability requires pooling of information across several assay runs. Simulation work indicates that use of empirical Bayes methods may afford considerable gain in efficiency; one must weigh this gain against practical considerations in the implementation of Bayesian techniques. We illustrate the methods discussed using a cell-based bioassay for the recombinant hormone relaxin.

Bayes Theorem↗

PubMed1992

A simple expression for quantifying bacterial chemotaxis using capillary assay data: application to the analysis of enhanced chemotactic responses from growth-limited cultures.

An individual cell-based mathematical model of Rivero et al. provides a framework for determining values of the chemotactic sensitivity coefficient chi 0, an intrinsic cell population parameter that characterizes the chemotactic response of bacterial populations. This coefficient can theoretically relate the swimming behavior of individual cells to the resulting migration of a bacterial population. When this model is applied to the commonly used capillary assay, an approximate solution can be obtained for a particular range of chemotactic strengths yielding a very simple analytical expression for estimating the value of chi 0, [formula: see text] from measurements of cell accumulation in the capillary, N, when attractant uptake is negligible. A0 and A infinity are the dimensionless attractant concentrations initially present at the mouth of the capillary and far into the capillary, respectively, which are scaled by Kd, the effective dissociation constant for receptor-attractant binding. D is the attractant diffusivity, and mu is the cell random motility coefficient. NRM is the cell accumulation in the capillary in the absence of an attractant gradient, from which mu can be determined independently as mu = (pi/4t)(NRM/pi r2bc)2, with r the capillary tube radius and bc the bacterial density initially in the chamber. When attractant uptake is significant, a slightly more involved procedure requiring a simple numerical integration becomes necessary. As an example, we apply this approach to quantitatively characterize, in terms of the chemotactic sensitivity coefficient chi 0, data from Terracciano indicating enhanced chemotactic responses of Escherichia coli to galactose when cultured under growth-limiting galactose levels in a chemostat.

Bacterial Physiological Phenomena↗

PubMed2026

Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors.

Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.

Hepatitis B virus↗

PubMed2024

Identification of potent pan-ephrin receptor kinase inhibitors using DNA-encoded chemistry technology.

EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that EPHA2 and EPHA4 expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (Ki: 4.0 nM) and EPHA4 (Ki: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC50: 461 nM) and EPHA4 (IC50: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (Ki: 0.13 nM) and EPHA4 (Ki: 0.38 nM) with excellent cell-based potency EPHA2 (IC50: 8.0 nM) and EPHA4 (IC50: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1β-mediated expression of prostaglandin synthase 2 (PTGS2). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.

Humans↗

PubMedDate not supplied

Detection of sodium channel toxins: directed cytotoxicity assays of purified ciguatoxins, brevetoxins, saxitoxins, and seafood extracts.

Neuroblastoma cells in culture were used to detect sodium channel-specific marine toxins based on an end-point determination of mitochondrial dehydrogenase activity. The assay responds in a dose-dependent manner to ciguatoxins, brevetoxins, and saxitoxins, and delineates the toxic activity as either sodium channel enhancing or sodium channel blocking. The assay responds rapidly to sodium channel activating toxins, allowing dose dependent detection in 4 to 6 h. Brevetoxins can be detected at 250 pg, and purified ciguatoxins are detected in the low picogram and subpicogram levels. The results obtained from cell bioassay of ciguatoxic finfish extracts correlates with those obtained from mouse bioassays. Sodium channel blocking toxins can also be detected with an approximate sensitivity of 20 pg in 24 to 48 h. This cell-based technique is simple, sensitive, demonstrates potential as an alternative to animal testing for sodium channel activating and blocking toxins, and can be automated.

Animals↗

PubMed2027

Screening of Antiviral Agents Against CHIKV Using Reporter Virus.

Chikungunya virus (CHIKV) causes a disease characterized by chronic musculoskeletal inflammation for which specific antivirals are not yet available. Currently, a supportive therapy to alleviate fever and pain is used, but it does not limit viral replication or the persistence of chronic arthritis symptoms. Thus, the identification and development of new active molecules against CHIKV is urgently needed. Here, we present a cell-based methodology that enables the implementation of a rapid and cost-effective strategy for high- and medium-throughput screening (HTS) of compounds, including repurposed drugs or novel molecules. This methodology allows for the identification of novel antiviral hits with a good activity and selectivity profile against CHIKV.

Antiviral Agents↗

PubMed2025

Systematic discovery of CRISPR-boosted CAR T cell immunotherapies.

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction remains a common cause of treatment failure1. Here we present CELLFIE, a CRISPR screening platform for enhancing CAR T cells across multiple clinical objectives. We performed genome-wide screens in human primary CAR T cells, with readouts capturing key aspects of T cell biology, including proliferation, target cell recognition, activation, apoptosis and fratricide, and exhaustion. Screening hits were prioritized using a new in vivo CROP-seq2 method in a xenograft model of human leukaemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered that RHOG knockout is a potent and unexpected CAR T cell enhancer, both individually and together with FAS knockout, which was validated across multiple in vivo models, CAR designs and sample donors, and in patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify synergistic gene pairs and saturation base-editing screens to characterize RHOG variants. In summary, we discovered, validated and biologically characterized CRISPR-boosted CAR T cells that outperform standard CAR T cells in widely used benchmarks, establishing a foundational resource for optimizing cell-based immunotherapies.

Humans↗

PubMed1995

Concept and progress in the development of RGD-containing peptide pharmaceuticals.

The cell adhesion domain, arginine-glycine-aspartic acid (RGD), has been incorporated into synthetic peptides to perform either of two modes of drug action, antagonist or agonist. Short, conformationally constrained peptides have been developed as antagonists for the platelet membrane glycoprotein complex, the integrin alpha IIb beta 3, using cell-based and integrin-based assays. In combination with a comparative molecular modeling study, these results have helped identify common conformational elements in the pharmacophore of this class of molecules. Peptides are presented that are highly potent, integrin specific, and that possess reduced pharmacological side effects. Also presented is the development of a peptide that modifies, noncovalently, the surfaces of a wide variety of synthetic materials used in medical implants. The agonist activity of [corrected] this molecule is evident from its ability to stimulate cell attachment on these surfaces. This is shown to translate into an in vivo activity of faster and more complete tissue integration, and a reduction in foreign body response.

Amino Acid Sequence↗

PubMed2026

Gata3 dosage governs primitive endoderm versus trophectoderm specification in embryonic stem cells.

Transcription factor (TF) dosage represents an overlooked aspect of developmental regulation. While Gata3 has traditionally been viewed as a determinant of trophectoderm (TE), its potential role in primitive endoderm (PE) has remained unclear. Here, we demonstrate that Gata3 functions as a dosage-sensitive regulator directing mutually exclusive lineage programs in mouse embryonic stem (ES) cells. Low levels of Gata3 (Gata3-L) promote PE-like transcriptional states, while high levels (Gata3-H) drive TE identity by rapidly repressing pluripotency and inducing TE markers. Genome-wide binding analysis reveals a dose-dependent redistribution of Gata3 across enhancers, with chromatin engagement consistent with pioneer factor-like activity. Functional 3D blastoid assays combined with single-cell transcriptomics further establish that Gata3 dosage alone is sufficient to instruct the spatial segregation of PE- versus TE-like compartments. These findings redefine Gata3 not merely as a TE determinant but as a central dosage-sensitive switch in lineage specification. More broadly, our results position TF dosage as a fundamental regulatory parameter that integrates enhancer selection, chromatin engagement, and spatial patterning, providing new opportunities to refine stem cell-based models and engineer developmental outcomes.

GATA3 Transcription Factor↗

PubMed2024

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

Humans↗

PubMed1994

Pharmacological modulation of endothelial cell-associated adhesion molecule expression: implications for future treatment of dermatological diseases.

Skin diseases with an inflammatory component, regardless of their etiology, are characterized at some point by the extravasation and subsequent infiltration of leukocytes into the dermal and/or epidermal compartments. This trafficking pattern is determined by a complex series of events whereby the leukocytes interact with cell adhesion molecules (CAM), particularly those induced on endothelial cells following activation with various inflammatory mediators. Vascular CAMs belonging to the selectin family (i.e., P-selectin and E-selectin) are thought to mediate early and reversible events involving leukocyte rolling and margination along the lumenal surface of microvascular cells (post-capillary venules). Certain members of the immunoglobulin supergene family (i.e., VCAM-1 and ICAM-1) regulate later and irreversible steps which lead to firm attachment and subsequent diapedesis of leukocytes. Accumulating evidence suggests that if one blocks the ligand-binding sites between leukocytes and endothelial cells, or inhibits vascular CAM expression, hematopoietic cell extravasation and progressive inflammatory events can be greatly diminished. To identify such inhibitors we developed a cell-based Elisa using the human microvascular cell line HMEC-1. As reported in the present paper, this approach yielded a naturally-occurring, low molecular weight compound which potently inhibits cytokine-induced adhesion molecule expression on cultured endothelial cells, without modulating "house-keeping" proteins.

Cell Adhesion Molecules↗

PubMed1986

Determination of ABO blood group zygosity by an antiglobulin rosetting technique and cell-based enzyme immunoassay.

Two methods were employed to determine the zygosity of A and B red cells, the rosetting antiglobulin test and the enzyme-linked immunoadsorbent assay (ELISA) technique. By means of the rosetting antiglobulin test, clear differences between AA and AO as well as BB and BO could be shown; however, this method could not define the variation in the amount of the H antigen as a complimentary means of differentiating AA from AO and BB from BO. The ELISA test could quantitatively estimate the level of expression of H antigen, and using anti-A and anti-B reagents in a simultaneous assay, a specific pattern could be generated for each phenotype. Both rosette and ELISA approaches offer easy and inexpensive means of determining zygosity within the ABO system for use in paternity exclusion.

ABO Blood-Group System↗

PubMed1994

C5a-induced expression of P-selectin in endothelial cells.

Human umbilical vein endothelial cells have recently been shown to respond to C5a with increases in intracellular Ca2+, production of D-myo-inositol 1,4,5-triphosphate and superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and endothelial cells in the acute inflammatory response.

Cell Adhesion↗

PubMed1995

Cytokine-mobilized peripheral blood CD34+Thy-1+Lin- human hematopoietic stem cells as target cells for transplantation-based gene therapy.

Gene-therapy of blood-borne disorders may be best achieved using hematopoietic stem cells (HSC) which have extensive self renewal potential as well as multilineage repopulating potential as a cellular target. The human HSC, which is CD34+Thy-1+Lin- has been isolated from fetal, adult bone marrow and cytokine-mobilized peripheral blood (MPB) (1-3). Results presented in this study show that the degree of mobilization of HSC into peripheral blood of cancer patients is highly variable and that the combined use of high dose chemotherapy and GM-CSF as a mobilization strategy is superior to the use of G-CSF with regard to the mobilization of true HSC. A multistep cell isolation procedure has been developed which utilizes high speed flow-cytometric cell sorting and allows the isolation of sufficient numbers of HSC from MPB to permit their use as an hematopoietic graft for clinical transplantation. Hematopoietic stem cells isolated from MPB are capable of self-renewal and differentiation into multiple hematopoietic lineages as shown by their behavior in both in vitro and in vivo assays. Mobilized PB mononuclear cells isolated from cancer patients are frequently contaminated with tumor cells. Using this cell isolation procedure, HSC preparations from patients with multiple myeloma have been created with greatly reduced tumor cell burdens. These CD34+Thy-1+Lin- cells are capable of being stably transduced at high efficiency (32-75%) by co-culture on a cell line producing recombinant retroviruses containing the neomycin-resistant gene. These HSC cell populations are likely ideal targets for hematopoietic cell-based gene therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗