Target/Analyte:
RO

Short Name RO
Description Metabolite from Sofosubuvir

Method/Kits with this Target/Analyte
Method/Kit Description
mCherry 587/610 Biosensor
Lonza AdipoRed Assay Reagent (Lonza: PT-7009) The AdipoRed Assay Reagent is designed for assessing the effect of compounds on the differentiation of preadipocytes or on lipid utilization in mature adipocytes. The lipophilic AdipoRed Assay Reagent specifically partitions into the fat droplets of differentiated adipocytes and fluoresces at 572 nm. The assay quantifies the accumulation of intracellular triglycerides. (Lonza)
Progesterone ELISA Kit Cayman's Progesterone ELISA Kit is a competitive assay that can be used for quantification of progesterone in plasma and other sample matrices. The assayhas a range from 7.8-1,000 pg/ml and a sensitivity (80% B/B) of approximately 10 pg/ml.
Flow Cytometry Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
nuc-GFP cell proliferation quantification of GFP nuclei in primary melanoma cells
QuantiChrom Urea Assay Kit (QuantiChrom: DIUR-100) For quantitative determination of urea and evaluation of drug effects on urea metabolism.
Urea Nitrogen (BUN) Colorimetric Detection Kit The Urea Nitrogen (BUN) Detection research-use-only kit is a colorimetric assay designed for the quantification and detection of urea nitrogen in serum, plasma, urine, saliva and tissue culture media samples. This complete, ready-to-use kit includes clear 96-well plate(s), urea nitrogen standard, and other components to perform the assay. A 96-well microplate reader capable of reading optical absorption at 450 nm is required for use of this kit. https://www.thermofisher.com/order/catalog/product/EIABUN#/EIABUN
DHE Reactive oxygen species chemical probe
Human alpha-Fetoprotein Quantikine ELISA Kit (R&D System; DAFP00) The Quantikine Human AFP immunoassay is a 4.5 hour solid phase ELISA designed to measure human AFP in cell culture supernates, serum, and plasma. https://www.rndsystems.com/products/human-alpha-fetoprotein-quantikine-elisa-kit_dafp00
Luminex MAGPIX Masurement of secreted protein
Thermo Scientific™ Pierce™ BCA Protein Assay Kit Green-to-blue (A562nm), precise, detergent-compatible assay reagent to measure total protein concentration vs. protein standard.
CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega: G7890, G7891, G7892) Progema LDH measure and cytotoxicity assay
DCFDA Cell permeable fluorescent and chemiluminescent probes. 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most widely used techniques for directly measuring the redox state and ROS of a cell.
LDH-Glo™ Cytotoxicity Assay The LDH-Glo™ Cytotoxicity Assay is a bioluminescent plate-based assay for quantifying lactate dehydrogenase (LDH) release into the culture medium upon plasma membrane damage. The bioluminescent detection is more sensitive than colorimetric or fluorescent methods, allowing accurate detection of LDH from a small number of cells, including primary cells and 3D cell cultures.
Luminex xMAP A bead-based multiplexed immunoassay system in a microplate format
Cell Proliferation Kit II (XTT) (Sigma: 11465015001) The Cell Proliferation Kit II (XTT) is a colorimetric assay for the nonradioactive quantification of cellular proliferation, viability, and cytotoxicity. Sample material is either adherent or suspension cells cultured in 96-well microplates. In contrast to MTT, the cleavage product of XTT is soluble in water; therefore, a solubilization step is not required. The tetrazolium salt XTT is cleaved to formazan by a complex cellular mechanism. This bioreduction occurs in viable cells only, and is related to NAD(P)H production through glycolysis. Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture. (Sigma)
Human ALT ELISA Kit (ab234578) Human ALT ELISA Kit (ab234578)
Human Bone Panel I Kit (MSD: K15146C) The Human Bone Marker Panel I Kit provides assay-specific components for the quantitative determination of both natural and recombinant human osteoprotegerin (OPGN), sclerostin (SOST), and alkaline phosphatase (ALP) in serum and plasma
Mass spectrometry Mass spectrometry
RT-PCR (Applied Biosystems: StepOnePlus and SYBR Green Reaction Mix) Real-time PCR noted in Tuan work. RT-PCR (Applied Biosystems: StepOnePlus and SYBR Green Reaction Mix) Lin et al. Mol. Pharmaceutics 2014) The SYBR Green I dye chemistry uses Applied Biosystems™ SYBR™ Green I dye, a highly specific, double-stranded DNA binding dye, to detect PCR product as it accumulates during PCR cycles. The most important difference between the TaqMan and SYBR Green I dye chemistries is that the SYBR Green I dye chemistry will detect all double-stranded DNA, including non-specific reaction products. A well-optimized reaction is therefore essential for accurate results.
Protein Fluorescence (DAPI, Texas Red) with Quantification (ImageJ, AngioTool) Quantification of an image with DAPI and Texas Read stains for vascular and tumor targets in the UC Irvine VMT model.
Fluorescence (490/525) Direct read or quantification of FITC (normally)
TGF-B1 ELISA ELISA to detect human TGF-B1
Cytogenetic analysis Giemsa banded (G-banded) karyotyping analysis Cytogenetic analysis Giemsa banded (G-banded) karyotyping analysis was performed for the chromosome analysis of immortalized cells. Cell samples were prepared when cells were in log phase, undergoing active division. The day before the collection, the cells were seeded at ∼40% of confluency in T75 flasks. AEC: 1,400,000/T75; AMC and CMC: 850,000; CTC: 1,200,000; and DEC: 700,000 cells were seeded to T75 flasks overnight. The next day, the cells were treated with 10 μL of KaryoMAX™ Colcemid™ (Gibco™, Cat. No: 15210040, 10 ug/mL)/10 mL complete media for 1 h in a 37°C, 5% CO2 incubator. Then, all cells (including floating cells) were collected and centrifuged at 500 rpm for 7 min at RT. The supernatants were carefully removed, and the pellets were resuspended in 8 mL 0.075 M KCL hypotonic solution for 7 min at RT, then centrifuged again at 500 rpm for 7 min at RT. The supernatants were carefully removed, and the pellets were slowly resuspended in fixation solution (3:1, methanol: acetic acid) for 15 min at RT. The fixative washing step was repeated two times. Then, the cells were stored in a fixative at 4°C until being shipping to Creative Bioarray LLC., Shirley, NY, USA, where G-banded karyotyping services were conducted. In some cases, live cells were shipped at 40% confluency in the Petaka G3™ Cell Culture Cassette (Gemini Bio, West Sacramento, CA, Cat. No: LTPTK25) at RT. Cytogenetic analysis was performed on 20 G-banded metaphase spreads for each cell line.
BUN Liquid Reagent (Stanbio Laboratory: SB-0580-250) Features of Stanbio Urea Nitrogen (BUN) Test: Diacetylmonoxime Methodology Ready-to-use liquid reagents Linear to 80 mg/dL Read @ 520 nm Standard included
BODIPY 581/591 C11-Lipid Peroxidation Sensor Measure lipid peroxidation with BODIPY C11 sensor
HCS LipidTOX Red Neutral Lipid Stain (Thermo: H34476) The intracellular accumulation of neutral lipids, steatosis, is often triggered by drugs that affect the metabolism of fatty acids and/or neutral lipids. HCS LipidTOX™ Red neutral lipid stain was developed to characterize the potentially toxic effects of compounds on lipid metabolism in mammalian cell lines. The LipidTOX™ neutral lipid stain has an extremely high affinity for neutral lipid droplets and can be detected by fluorescence microscopy or an HCS reader. This probe is compatible with the HCS LipidTOX™ phospholipidosis detection reagents (H34350, H34351). HCS LipidTOX™ neutral lipid stains can also be used to monitor the formation and differentiation of adipocytes, a process called adipogenesis.
CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega: G1780) The CytoTox 96® Non-Radioactive Cytotoxicity Assay is a colorimetric alternative to 51Cr release cytotoxicity assays. The assay measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured with a 30-minute coupled enzymatic assay, which results in conversion of a tetrazolium salt (INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells. Visible wavelength absorbance data are collected using a standard 96-well plate reader.
LipidTOX staining Invitrogen™ HCS LipidTOX™ Deep Red Neutral Lipid Stain, for cellular imaging Catalog number: H34477
Edu Click-iT and CD markers staining Cell proliferation, Cell viability and Myeloid Cell lineage differentiation Surface markers
Enolase 2 (Neuron Specific Enolase) Antibody Set (R-PLEX) Human Enolase 2 / Neuron Specific Enolase (NSE) Antibody Set Features Measures human Enolase 2 in Serum, Plasma, CSF Dynamic range: 8.7 - 100,000 pg/mL Median LLOD: 8.7 pg/mL
Single-Use RED Plate with Inserts (Thermo Scientific: 2034.6) and ICP-MS Single-Use RED Plate with Inserts (Thermo Scientific: 2034.6) followed by mass spectrometry for quantification of compound and determination of: % Free = (Concentration buffer chamber/Concentration plasma chamber) × 100% and % Bound = 100% - % Free.
Quantitative PCR (ThermoFisher) QuantStudio 12K Flex Real-Time PCR System with SYBR Green Master Mix
Fluorescence microscopy (ROS) Video microscopy imaging of reactive oxygen species produced in the cell via measuring the mean intensity values of DHR. Quantification of the images were performed with an in house software (JEX) developed at the Beebe lab at UW Madison.
Testosterone, IM/MS Testosterone is metabolized by human Cyp 2C9, Cyp 2C19 and Cyp 3A4. The Cyp 3A4 isoenzyme is solely responsible for the human testosterone metabolite 6β-Hydroxytestosterone. In this protocol the level of 6β-Hydroxytestosterone and testosterone found in the liver efflux media is used as a monitor of liver induction or inhibition.
Human NT-proBNP (MSD: F214I-3) Measures human N-terminal pro-brain (or B-type) natriuretic peptide, NT-proBNP on the MSD R-PLEX platform
LC ms/ms mass spec
Immunocytochemistry and immunofluorescence protocol Immunocytochemistry (ICC) combines anatomical, immunological and biochemical techniques to image discrete components in tissues by using appropriately-labeled antibodies to bind specifically to their target antigens in situ.
Western Blot An immunoassay that detects specific proteins in the laboratory samples
McAleer 2019 Image Quantification (ImageJ) The imaging processing was developed for and performed in ImageJ (51) and incorporated a contrast enhancement and manually directed brightness thresholding operation, followed by an area determination occupied by the cells. A series of five focal plane images were collected and analyzed for each data point, and the cell density among the five images was averaged.
Human alpha-Fetoprotein DuoSet (R&D Systems: DY1369) INTENDED USE: For the development of sandwich ELISAs to measure natural and recombinant human alpha Fetoprotein (α-Fetoprotein). The Reagent Diluent recommended may be suitable for most cell culture supernate, serum, and plasma samples. This kit contains sufficient materials to run ELISAs on at least fifteen 96 well plates.
LC-MS/MS Determination of analyte concentration by LC-MS/MS
Human Prolactin Antibody R&D Systems, Cat. No: AF682
ProcartaPlex assay Cytokine detection assay
HPLC-MS As the name suggest the instrumentation comprises a high performance liquid chromatograph (HPLC) attached, via a suitable interface, to a mass spectrometer (MS). The primary advantage HPLC/MS has over GC/MS is that it is capable of analysing a much wider range of components.
Human cardiac Troponin T (F214L-3) Measures human cardiac troponin T on the MSD R-PLEX platform
Testosterone ELISA (mouse/rat) Mouse/Rat Testosterone ELISA is for the quantitative determination of Testosterone concentration in mouse/rat serum or plasma.
Water Contact Angle Measure the sessile drop water contact angle
Corrosivity Assessment | PrestoBlue Cell Viability (ThermoFisher: A13261, A13262) An assessment of compound corrosion based on processing data from the PrestoBlue Cell Viability assay.
Microscopy - Fluorescence Imaging with some unspecified number of fluorescent marker and/or stains.
Propidium Iodide/Hoechst 33342 PI Stain
Pierce LDH Cytotoxicity Assay Kit (ThermoFisher: 88953, 88954) LDH Assay (NADPH?)
Fluorescence microscopy (Hoechst) Video microscopy imaging of Hoechst nuclear staining to quantify the formation of neutrophil extracellular traps. Quantification of the images were performed with an in house software (JEX) developed at the Beebe lab at UW Madison.
CytC Biosensor, Image Algorithm In healthy cells CytC is localized to the mitochondria. On induction of Apoptosis, CytC is released from the mitochondria. The biosensor consists of a fluorescent tagged CytC, that upon release becomes more diffuse and dimmer.
Cytotoxicity Detection Kit (Roche: 11644793001) Colorimetric assay for the quantification of cell death and cell lysis, based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant.
Hematoxylin and Eosin (H&E) Staining (Baylor College of Medicine Protocol) Hematoxylin & Eosin Stain (H&E) for imaging of the nucleus, cytoplasm, and extracellular proteins. This H&E staining follows the protocol created by the Baylor College of Medicine for staining samples from their Bone MPS model.
Single-Use RED Plate with Inserts (Thermo Scientific: 2034.6) and LC-MS Single-Use RED Plate with Inserts (Thermo Scientific: 2034.6) followed by mass spectrometry for quantification of compound and determination of: % Free = (Concentration buffer chamber/Concentration plasma chamber) × 100% and % Bound = 100% - % Free.
Immunofluorescence indirect or direct immunofluorescence staining
Unspecified Not specified by the user
Confocal Microscopy Leica SP5 Inverted Confocal Microscope. Excitation frequencies used: 405, 488, 633
Urea assay kit (Sigma-Aldrich MAK006) Urea assay kit (Sigma-Aldrich MAK006)
Absorbance Absorbance of a sample, measured by plate reader.
Cholesterol Assay Kit (Fluorometric Assay) - HDL and LDL/VLDL (ab65390) Detection of FREE cholesterol and TOTAL cholesterol from two independent replicates from three different fractions of a single sample. Total sample, HDL fraction, and LDL/VLDL fraction; fractions separated by centrifugation with PRECIPITATION buffer. Cholesterol esterase hydrolyzes cholesteryl ester to free cholesterol. OxiRed Probe detects all free cholesterol. FREE cholesterol samples don't get exposed to esterase. Samples are measured by Fluorescence in a plate reader Ex/Em = 535/587 nm. VLDL is calculated by subtracting the TOTAL cholesterol from the FREE cholesterol.
Pars Azmun Commercial assays sold by Iranian company Pars Azmun
MILLIPLEX MAP Human Kidney Injury Magnetic Bead Panel 1 - Toxicity Multiplex Assay The analytes available for this multiplex kit are: Calbindin, Collagen IV, Fatty Acid Binding Protein 1 (FABP1), GSTα, IP-10, Kidney Injury Molecule-1 (KIM-1), Osteoactivin, Renin, TFF-3, TIMP-1.
Elise Procollagen Type1A1 measure secretome collagen 1A1 fragments
LDH Assay Kit (abcam: ab102526) Lactate Dehydrogenase (LDH) Assay Kit (Colorimetric) (ab102526) quantifies LDH activity in a variety of biological samples such as serum or plasma, cells, culture medium and fermentation. In this assay LDH reduces NAD to NADH, which then interacts with a specific probe to produce a color (OD max = 450 nm). The kit can detect 1 - 100 mU/mL of LDH directly in samples. The assay is quick, convenient, and sensitive.
GFAP Antibody Set, Human (R-PLEX) Measures human Glial Fibrillary Associate Protein in Plasma Dynamic range: 63 - 500,000 pg/mL Median LLOD: 63 pg/mL
RapidFire-MS The RapidFire 365 High-Throughput Mass Spectrometry System significantly increases the throughput of LC/MS analyses by replacing HPLC with automated solid phase extraction (SPE). An integrated robot manages reusable SPE cartridges, which are available in a variety of chemistries to optimize selectivity. The RapidFire 365 is a high speed autosampler that can be integrated with Agilent’s Triple Quadrupole, Time-of-Flight, and Quadrupole Time-of-Flight mass spectrometers. RapidFire enables automated high-capacity sample analysis in a wide range of applications, even in biological matrices. The technology provides a more successful approach to HTS of challenging drug targets and a 10x faster throughput for MS-based analyses like HT-ADME discovery assays. This end to end solution delivers shorter time to results, reduced operational costs and automated high-capacity sample processing.
Immunofluorescence indirect or direct immunofluorescence staining
Protein Fluorescence (DAPI, Texas Red) Unaltered image with DAPI and Texas Read stains for vascular and tumor targets in the UC Irvine VMT model.
Microscopy - Confocal 405, 488, 633
Protein Fluorescence (DAPI, Texas Red) with Threshold (ImageJ) Thresholded image (using ImageJ) with DAPI and Texas Read stains for vascular and tumor targets in the UC Irvine VMT model.
Lonza AdipoRed Assay Reagent (Lonza: PT-7009), Hoechst 33342 Combined assay and stain. The AdipoRedTM Assay Reagent is designed for assessing the effect of compounds on the differentiation of preadipocytes or on lipid utilization in mature adipocytes. The lipophilic AdipoRedTM Assay Reagent specifically partitions into the fat droplets of differentiated adipocytes and fluoresces at 572 nm. This objective, high-throughput, homogeneous fluorescence-based assay quantifies the accumulation of intracellular triglycerides and provides significant advantages to drug discovery efforts in the field of obesity and diabetes. It is more sensitive than other methods such as the Oil Red O assay, and is much faster and easier than Northern and Western blots (https://bioscience.lonza.com/lonza_bs/US/en/Cell-analysis/p/000000000000186733/AdipoRed-Assay-Reagent%2C-20-mL). And Hoechst 33342 to see the nucleus.
Protein Fluorescence (mCherry) mCherry protein fluorescence used to examine cells by imaging.
Immunocytochemistry and immunofluorescence protocol (DAPI, FITC, Cy5) Immunocytochemistry (Cy5, DAPI, FITC) used for imaging and observation of HO-1, DNA, OAT4.
Hematoxylin and Eosin (H&E) Staining (Baylor College of Medicine Protocol) Hematoxylin & Eosin Stain (H&E) for imaging of the nucleus, cytoplasm, and extracellular proteins. This H&E staining follows the protocol created by the Baylor College of Medicine for staining samples from their Bone MPS model.
Hematoxylin and Eosin (H&E) Staining Hematoxylin & Eosin Stain (H&E) for imaging of the nucleus, cytoplasm, and extracellular proteins. This has no specific protocol for the staining
Biliary efflux assay Using FDA as a substrate, measuring fluroscein excretion

Studies Utilizing this Target/Analyte
Study Start Date Study Types Description
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