acid-phosphatase and resazurin

Mainstream adoption of physiologically relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price, and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily available reagents and open-source software to analyze spheroid volume, metabolism, and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination-for both single images and images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyze plate uniformity (such as edge effects and systematic seeding errors), detect outliers, and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time.

Topics: Acid Phosphatase; Brain; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Humans; Image Processing, Computer-Assisted; Indicators and Reagents; Oxazines; Reproducibility of Results; Sensitivity and Specificity; Software; Spheroids, Cellular; Time Factors; Xanthenes

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

Topics: Acid Phosphatase; Antineoplastic Agents; Biological Assay; Brain; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Drug Screening Assays, Antitumor; Humans; Oxazines; Reproducibility of Results; Spheroids, Cellular; Stem Cells; Tumor Cells, Cultured; Xanthenes

The motility assay is the current gold standard for evaluating drug effects on hookworm larvae and adults, however, among other drawbacks the assay is time consuming, and prone to individual subjectivity. We evaluated six alternative in vitro assays, namely the feeding inhibition assay, the colourimetric AlamarBlue®, MTT formazan and acid phosphatase activity assays, as well as isothermal calorimetry and the xCELLigence System using Ancylostoma ceylanicum third-stage larvae, stimulated third-stage larvae and adults. The performances of the assays were compared to the motility assay using three standard drugs: albendazole, levamisole and ivermectin (100-1 μg/ml). None of the assays investigated offered an advantage over the motility assay, because they were all inapplicable to third-stage larvae, which were presumably metabolically and physically too inactive. Among all assays tested the xCELLigence System performed best on adult worms as the test was accurate, simple, required a minimal number of worms and offered the possibility for conducting a medium-throughput screening.

Topics: Acid Phosphatase; Albendazole; Ancylostoma; Animals; Anthelmintics; Calorimetry; Cell Migration Assays; Colorimetry; Drug Resistance; Formazans; High-Throughput Screening Assays; Humans; Indicators and Reagents; Ivermectin; Larva; Levamisole; Oxazines; Parasitic Sensitivity Tests; Sensitivity and Specificity; Tetrazolium Salts; Xanthenes

Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhibition of bone resorption without inhibition of bone formation. We investigated the role of acidification in human osteoclastic resorption and life span in vitro using inhibitors of chloride channels (NS5818/NS3696), the proton pump (bafilomycin) and cathepsin K. We found that bafilomycin and NS5818 dose dependently inhibited acidification of the osteoclastic resorption compartment and bone resorption. Inhibition of bone resorption by inhibition of acidification, but not cathepsin K inhibition, augmented osteoclast survival, which resulted in a 150 to 300% increase in osteoclasts compared to controls. We investigated the effect of inhibition of osteoclastic acidification in vivo by using the rat ovariectomy model with twice daily oral dosing of NS3696 at 50 mg/kg for 6 weeks. We observed a 60% decrease in resorption (DPYR), increased tartrate-resistant acid phosphatase levels, and no effect on bone formation evaluated by osteocalcin. We speculate that attenuated acidification inhibits dissolution of the inorganic phase of bone and results in an increased number of nonresorbing osteoclasts that are responsible for the coupling to normal bone formation. Thus, we suggest that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.

Topics: Acid Phosphatase; Adult; Animals; Bone and Bones; Bone Density; Bone Resorption; Cathepsin K; Cathepsins; Cells, Cultured; Chloride Channels; Dose-Response Relationship, Drug; Family Health; Female; Humans; Immunohistochemistry; Indicators and Reagents; Isoenzymes; Macrolides; Macrophage Colony-Stimulating Factor; Macrophages; Male; Middle Aged; Models, Biological; Mutation; Osteoclasts; Osteopetrosis; Ovary; Oxazines; Phenotype; Phenylurea Compounds; Rats; Rats, Sprague-Dawley; Tartrate-Resistant Acid Phosphatase; Tetrazoles; Time Factors; Xanthenes

Cultured microglial cells usually exhibit ameboid morphology and peripheral macrophage-like properties, which are distinct from those observed in the normal mature brain. This might be caused by the inappropriate culture of microglial cells in high concentrations (approximately 200-400 microM) of Gly and Ser, although the concentrations of the amino acids in extracellular spaces of the brain parenchyma are quite low (approximately 5 microM). In the present study, we focused on the concentration-dependent effects of glycine (Gly) and serine (Ser) on microglial morphology and function. Under Gly/Ser-free and serum-free condition, the majority of rat microglial cells displayed round morphology, whereas in the presence of 5 microM Gly and 25 microM Ser, which correspond to the concentrations of Gly and Ser in the cerebrospinal fluid, they extended multiple branched processes and formed clusters of rough endoplasmic reticulum. On the other hand, Gly and Ser did not affect morphology of astrocytes. The viability of microglia was not affected by the changes in the concentrations of Gly and Ser. Metabolic activity, activities of acid phosphatase and inducible nitric oxide synthase, and superoxide anion (O2-) generation were all strongly suppressed in Gly/Ser-free medium or in medium containing physiological concentrations of both amino acids. Such activities were all enhanced in harmony with increases in the concentrations of Gly and Ser. Thus, microglial cells cultured in Gly/Ser-free medium, even though exhibiting ameboid morphology, appears to be in the functionally resting state. Furthermore, once the resting state was achieved, the microglial cells remained inactive even after the subsequent 24 h culture in serum-supplemented medium containing 400 microM of both amino acids. The medium conditioned by microglial cells that were cultured in the presence of 400 microM of Gly and Ser was toxic to cortical neurons, whereas the microglia-conditioned medium obtained in the absence of both amino acids facilitated the survival of cortical neurons. Therefore, microglial cells in the resting state, which was induced in the Gly/Ser-free condition, are likely to support neurons. Microglial cells could ramify on glass coverslips coated with astrocyte-derived extracellular matrix or on coverslips coated thinly with fibronectin and/or laminin even under the Gly/Ser-free condition. The ramified cells as induced in this way kept suppressed O2- generating activity. These findi

Topics: Acid Phosphatase; Animals; Cells, Cultured; Coloring Agents; Culture Media; Extracellular Matrix Proteins; Fibronectins; Glycine; Laminin; Macrophage Activation; Microglia; Microscopy, Electron; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxazines; Oxygen Consumption; Rats; Serine; Superoxides; Trypan Blue; Xanthenes