UCL Drug discovery group application note. Author: Dr.

UCL Drug discovery group application note. Author: Dr.

UCL Drug discovery group application note. Author: Dr. Ben Allsop Cell toxicity assays *Data in this presentation are owned by the UCL DDG and are not for unauthorised reproduction* School of Pharmacy 29-39 Brunswick square WC1N 1AX Translational Research office Types of cell death Cell death is a major feature of disease and an important part of developmental biology. Death may occur in response to external injury, internal cues or programmed redundancy. Necrosis (extrinsic injury induced cell death) Cytoplasmic swelling followed by rupture of the plasma membrane.

Death receptor activation (Fas, TRAIL-Receptor etc.) Release of DNA from the nucleus. Mitochondrial swelling and decreased ATP production. Apoptosis (cell suicide) Membrane blebbing and organelle compartmentalisation Cytochrome C binds and activates procaspase-9 (Apoptosome) Eventually followed by necrotic cell death. Measuring cell toxicity Cell Viability CellTiter-glo (Promega) is an endpoint luminescence assay used to determine cell viability. This assay results in cell lysis and a signal proportional to the amount of ATP present in the well. The amount of ATP is directly proportional to the number of cells present. CellTiter-glo is a simple 1-step addition at a fixed timepoint. NB: Luminescence readouts are often O2 dependent and may have a reduced signal in

hypoxia. Cell death CellTox-Green (Promega) is green channel fluorescent assay able to directly measure cell death. This assay uses a non-cell permeable DNA binding flurophore, meaning it can directly measure the free-DNA of burst cells (Necrotic death). CellTox-Green is well tolerated and non-toxic, it can be used kinetically to measure cell death over hours or days. Kinetic measurement of cell death Measuring cell death throughout reperfusion injury in vitro Ischaemia- Understanding the kinetics of cell death following an insult can be very important to inform therapeutic interventions, as well as the underlying biology.

CellTox-Green enables kinetic measurement of cell death in cycles as short as 5min intervals (Fig. 1), or staggered over hours or days. This may be multiplexed with other kinetic dyes to maximise data output. For example - ROS, MPTP and Intracellular O2 dyes. Reading every 5 min for 24h causes little-to-no bleaching of the fluorophore. Cell tox green has been used at timepoints up to 72h in our lab. Fig 1: Rat myoblast cell death in response to 1h simulated ischaemiareperfusion injury. Kinetic measurement of chamber O2 (%) (Red) and cell death (blue) measured by CellTox-Green every 5min over a short ischaemiareperfusion protocol. Atmospheric gases were manipulated using Clariostar plate reader fitted with atmospheric control unit. Cell counting by automated immunofluorescence a) b)

Measuring cell death and DNA damage in response to olaparib treatment. Automated fluorescence microscopy with the Cytation (Biotek) of Hoechst 33342 staining allows for assaying nuclei following treatment. Multiples images per well of 96-well and 384-well plates can be stitched together to give whole-well data (Fig. 2a). Automated nuclei identification can be used as a measure of cell death (Fig. 2b). Multiplex with antibody staining (for example H2AX to H2AX to identify double stranded DNA breaks (Fig. 2c)) or endpoint mechanistic dyes (such as the MTMP dye Mitotraker Red) to investigate mechanisms of toxicity. c) Fig 2: Effect of Olaparib treatment in mesothelioma cells. Cancer cells were fixed with 4% PFA and stained with Hoechst 33324. 9

overlapping images per well were stitched together and deconvolved to form each image in (a). Automatic analysis counts cells and reports mean + standard deviation per sample (b). Representation of automated nuclei All assays can be performed in 96-well and 384-well format to increase throughput. All assays are suitable for both adherent and non-adherent cell culture. The DDG have several plate readers available for use. Easily perform synergy experiments between compound treatments (for example: test

compound + [Menadione]) Compound hit picking, serial dilution and cell treatments are automated to increase throughput and data reproducibility using the BRAVO (Agilent) liquid handling system. We are happy to discuss other assays not mentioned in this App note (for example the Annexin 5 Apoptosis/ Necrosis multiplex assay (Promega)). For questions and further information please contact: Richard Angell Drug discovery group lead; ([email protected]) Trevor Askwith Senior biologist; ([email protected]). Ben Allsop Biologist (Author); ([email protected]).

Recently Viewed Presentations



    Title: GEOMETRY Author: mciuser Last modified by: Iteach User Created Date: 3/13/2003 6:08:29 AM Document presentation format: On-screen Show (4:3) Company
  • Text Text Text Venn Diagram Guide Text Text

    Text Text Text Venn Diagram Guide Text Text

    Definition of Venn Diagram. P. Venn diagram is an illustration that uses overlapping or non-overlapping circles to show the relationship between a finite collection of sets. Where the circles overlap, items have a specified something in common. Some even refer...
  • 3.2- Attitude formation and measuring - Weebly

    3.2- Attitude formation and measuring - Weebly

    Aggression. Aggression is often linked back to attitude. In sport it is spoken about in both a positive and negative way. Being aggressive when defending is seen as positive but if it leads to injury of another player it will...
  • Diapositiva 1

    Diapositiva 1

    EL PROYECTO EDUCATIVO DEL PROGRAMA (PEP) Que es? El PEP es un documento que cada programa de la Universidad Cooperativa de Colombia debe elaborar con base al Acuerdo No. 002 de 2010.
  • Introduction - British Gypsum

    Introduction - British Gypsum

    Introduction to Weber Tile Fixing Products Product Range Weber's product range is split into 4 areas Preparation Fix Set Joint Preparation products These include a primer, levelling compounds and a tanking kit.
  • Meet Gmail - Valparaiso University

    Meet Gmail - Valparaiso University

    Survey Feedback. 81% of respondents were Satisfied or Very Satisfied with Gmail as an email client. 82% of respondents agreed that Gmail was an improvement over GroupWise. 84% of respondents rated the Gmail migration experience as
  • Review of Free Electron Theory - جامعة الملك سعود

    Review of Free Electron Theory - جامعة الملك سعود

    Physical model: In metallic state most loosely bound electrons some how become mobiles. These electrons behaves like classical particles generally we call it electron gas. Energy of the electron is determined by the temperature of metal. The electrons don't interact...
  • "It is a riddle, wrapped in a mystery, inside an enigma"*

    "It is a riddle, wrapped in a mystery, inside an enigma"*

    The Enigma transformation for each letter can be specified mathematically as a product of permutations. Assuming a three-rotor German Army/Air Force Enigma, let P denote the plugboard transformation, U denote that of the reflector, and L,M,R denote those of the...