De-risking drug discovery and development

Telephone:9818620

Email: dmbel.labservices@mbb.upd.edu.ph

Contact person:Marie Isabelle Viola (Lab Services Coordinator)

General Overview

The Laboratory Services arm of the Disease Molecular Biology and Epigenetics Laboratory (DMBEL) offers a suite of mostly cell-based assays to serve the needs of DOST-funded drug discovery projects as well as third-party clients.Bioactive hits from primary screens can be tested for drug-induced hepato-, nephro- and cardiotoxicity, which are responsible for high attrition rates in early-stage drug discovery. Confirmatory orthogonal and secondary assays are also available to eliminate artefactual drug bioactivities from primary screens due to pan-assay interference compounds and color effects. Most assays are in 96- or 384-well format and some are pathway-specific,thus providing clues on mechanism of action or the signaling cascade in which an extract/compound impinges. High-content imaging (using GE Healthcare’s IN Cell Analyzer 6000) is used in many of the assays, allowing more physiologically relevant readouts that are able to visualize subcellular and organelle phenomena.The medium-to-high throughput nature of HCI also affords multi-parametric information from each sample and even from individual cells, delivering different insights on a compound’s mode of action and/or non-specific effects early on.

Assays available:

In vitro toxicity assays

  • Hepatotoxicity Testing via Lactate Dehydrogenase Quantification
  • Nephrotoxicity Testing via Lactate Dehydrogenase Quantification
  • Cardiotoxicity Testing via Lactate Dehydrogenase Quantification
  • Fluorescence-based Cardiotoxicity Testing via Potassium Ion Channel Assay

Inflammation

  • Luminescence-based Assessment of COX-2 Transcriptional Activity
  • High-Content Imaging Fluorescence-based Detection of 5-LOX
  • High-Content Imaging-based Detection and Quantification of Reactive Oxygen Species by DCFDA Staining
  • High-Content Imaging-based Immunocytochemical Detection and Quantification of Prostaglandin E2
  • Expression Profile Analysis of Inflammation Markers by qRT-PCR

Diabetes

  • Luminescence-Based Glucose Uptake –GLOTM Assay

Hypercholesterolemia

  • High-Content Imaging-based Fluorescence Detection of Cholesterol Uptake via FilipinCytochemical Staining
  • High-Content Imaging-based Fluorescence Detection of LDL Uptake via BODIPY FL LDL Live Cell Staining

Hypertension

  • Determination of ACE Activity using Abz-FRK(Dnp)P-OH Substrate

Gout

  • Fluorescence-based Xanthine Oxidase Inhibition Assay
  • Fluorescence-based Uric Acid Quantification Assay

Obesity

  • Colorimetric Quantification of Lipid Droplet Accumulation via Oil Red O Staining
  • Colorimetric and FluorometricQuantification of Triglyceride Content

Cancer: Cell Proliferation

  • Luminescence-based Detection of ELK Pathway Activation
  • RealTime-GloTM Cell Viability Assay
  • Vybrant® MTT Cell Proliferation Assay
  • Cell Titer 96® Aqueous One Solution MTS Cell Proliferation Assay

Cancer: Apoptosis

  • Luminescence-based Detection of Caspase 3/7 Activity
  • Luminescence-based Detection of Caspase 8 Activity
  • Luminescence-based Detection of Early Apoptosis using the RealTime-GloTMAnnexin V Apoptosis and Necrosis Assay

Cancer: Migration and Invasion

  • Scratch Wound Healing Assay
  • Assessment of Tumor Cell Migratory Capacity via Boyden Chamber Assay
  • Expression profile analysis of EMT Markers by qRT-PCR
  • Expression profile analysis of EMT Markers by Western Blot

Cancer: Angiogenesis

  • High-Content Imaging Assisted Vessel Formation Assay

Cancer: Cytoskeletal Organization

  • Phalloidin Staining of F-actin Filaments

High-Content Imaging

  • Image Acquisition using IN Cell Analyzer 6000 High-Content Imaging System

Specialized Equipment Available:

High Content Imaging

  • IN Cell Analyzer 6000 (GE Healthcare)

Functionalities:

  • Time-lapse
  • Temperature, CO2 and humidity controls
  • Confocal option

Time-lapse microscope

  • IX83 (Olympus)

Functionalities:

  • Brightfield, fluorescence (TRITC/FITC/DAPI)
  • Time-lapse; motorized stage
  • Temperature, CO2 and humidity controls
  • Z-stack option

Multimode Readers

  • FluoSTAR Omega (BMG Labtech)

Functionalities:

  • Absorbance, Fluorescence, Luminescence detection modes
  • Bottom optics and multi-mode shaking; Built-in reagent injectors available
  • CLARIOstar (BMG Labtech)

Functionalities:

  • Absorbance, fluorescence, luminescence, polarized fluorescence, time resolved fluorescence and alpha-screen (Amplified Luminescent Proximity Homogeneous Assay) detection modes
  • Equipped with LVF MonochromatorTM technology (Linear Variable filters) for greater sensitivity of fluorescence and luminescence readings with adjustable bandwidths up to

100 nm

  • Top and bottom optics; Built-in reagent injectors available

Flow Cytometer

  • Attune NxT Flow Cytometer (Thermo Fisher Scientific)

Functionalities:

  • Multi-parametric single cell analysis with minimal compensation panels
  • Rapid processing and detection of multiple samples and of rare events
  • FSC, SSC, 14 fluorescent colors with PMTs, and user changeable detection methods are available for the following filters: Blue: 488 nm (50 mW), Violet: 405 nm (50 mW), Red:637 nm (100 mW) Yellow: 561 nm (50 mW)

Quantitative PCR

  • QuantStudio5 (Thermo Fisher Scientific)

Functionality:

  • Equipped with 96-well thermal blocks with 0.1mL well volume that can accommodate 3 independent temperature zones for PCR optimization (VeriFlex technology)
  • Runs can be monitored remotely through the Thermo Fisher Cloud

PCR Machines

  • C1000 Touch Thermal Cycler (Bio-Rad)

Functionality:

  • Dual block PCR machine capable of running 2 independent protocols simultaneously, with 48 PCR reactions in 0.2mL tubes per block
  • Equipped with gradient capability to accommodate 6 temperature zones
  • PCRmax Alpha Cycler 4 (Cole Parmer)

Functionality:

  • Quad block PCR machine capable of running 4 independent protocols simultaneously, with 96 PCR reactions in 0.2mL tubes per block
  • Equipped with gradient capability to accommodate at least 12 temperature zones

Nucleofector/Electroporator

  • Neon Transfection System (Thermo Fisher Scientific)

Functionality:

  • Specializes in highly efficient transfection of primary cells, stem cells and difficult-to-transfect cells

Chemidoc System

  • Chemidoc Touch Imaging System (Bio-Rad)

Functionality:

  • Capable of visualizing nucleic acid gels (Ethidium bromide, Gel Red, Sybr GREEN), protein gels, and western blots (Chemiluminescence, colorimetric, alexa 488, etc)

Automated cell counter

  • TC20 (Bio-Rad)
  • Countess II FL Automated Cell Counter (Thermo Fisher Scientific)