Trevigen s CometChip Platform (High Through-Put DNA Damage Analysis)

Transcription

1 Trevigen s CometChip Platform (High Through-Put DNA Damage Analysis) info@trevigen.com 8405 Helgerman Court, Gaithersburg MD 20817

2 Objectives Review Standard Comet Assay. Explain CometChip Platform. Demonstrate advantages of Trevigen Comet Analysis Software. Show validation of 4X image analysis.

3 The Comet Assay The Single Cell Gel Electrophoresis assay (SCGE, also known as comet assay) is a sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in It a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. The term "comet" refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet.

4 Principles of the Comet Assay Cells placed in LM Agarose Treat with Lysis solution Alkaline Electrophoresis and stain Large Fragments Small Fragments Immobilize cells on CometSlide Alkali Treatment

5 Typical Comet Data

6 The CometChip Platform The CometChip Platform consists of: CometChip Well Former Comet Electrophoresis System Comet Analysis Software CometChips Reagents CometChip

7 Trevigen s CometChip Platform: Reagents, Apparatus and Software CometChip Well Former CometChip Electrophoresis Unit with Power Supply CometChip Reagents Comet Analysis Software

8 CometChip provides high density data with minimal overlaps compared to older comet formats Older Comet Format CometChip

9 Trevigen Comet Analysis System (Analyze >500,000 comets per week) SCAN ANALYZE EDIT Wizard Driven Software

10 Free Demonstration at Booth 1728 Directions from Wizard Automatic Comet finding and Scoring Drag and Drop Images Study Name Review from Image Edit based on profile

11 Trevigen Comet Analysis System For the analysis of traditional comet data or CometChip Uses 4X images - requires fewer images Automatic Comet finding and scoring. No need to manually identify each comet. Easy to use and intuitive software. Easy data exports. Advantages w/ 96 well-cometchip (maximum 400 comets/well) ~150 comets per 4X image (camera/scope specific). Scan and analyze ~14,400 comets in < minutes. Minimal editing due to no over lapping comets.

12 Validation of 4X Imaging To date Comet analysis is based on the use of 10X images and frequently requires a software compatible camera. Trevigen platform uses a 4X image and is not camera specific. It was important to demonstrate that data generated from 4X images correlates with 10X images.

13 Software Validation Using Simulated Comets Simulated Comets The simulated comet images were designed to represent a range of damage levels and were constructed by using solid circular and elliptical shapes of selected intensity (grey scale) values to mimic the basic shapes and intensities of typical comets. These comet images were then convolved with a 15 X 15 smoothing filter to make the appearance of the simulated comets more realistic. These simulated comets were then arrayed in a single image in an arrangement that would make their order of processing by the program predictable (for comparison purposes.)

14 Excellent Correlation Between Expected and Program Generated Analysis of Simulated Comets PROGRAM Moment R² = PROGRAM % DNA in Tail R² = EXPECTED PROGRAM R² = Tail Length EXPECTED EXPECTED

15 Trevigen Analysis System Provide Comparable Results for 4X and 10X Images in Standard Neutral Comet Assays Jurkat Cells treated with increasing concentrations of Bleocin Each treatment was analyzed and averaged over 2 wells. Images were acquired with 4X and 10X objectives. 10X images were analyzed using Loats Automatic Comet Work Station. 4X images were analyzed using Trevigen Comet Analysis Software.

16 Trevigen Analysis System Provide Comparable Results for 4X and 10X Images in Standard Neutral Comet Assays 20 Moment 60 % DNA in Tail 4X Image R² = X Image 150 4X Image Tail Length R² = X Image 100 4X Image 50 0 R² = X Image

17 Trevigen Analysis System Provide Comparable Results for 4X and 10X Images in the Standard Alkaline Comet Assay Trevigen Alkaline Control Cells were run on a 20 well slide. Each treatment was analyzed and averaged over 5 wells. Images were acquired with 4X and 10X objectives. 10X images were analyzed using Loats Automatic Comet Work Station and Trevigen Software. 4X images were analyzed using Trevigen Comet Analysis Software.

18 Trevigen Analysis System Provide Comparable Results for 4X and 10X Images in the Standard Alkaline Comet Assay % DNA in Tail Comparisons Trevigen software (4X tiff) R² = Trevigen software (10X tiff) R² = Loats automated system (10X) Loats automated system (10X) 4X vs 10X tiff images: 1:1 correlation between Trevigen software and Loats automated system

19 Test Inter and Intra run variability with CometChip Platform Design Electrophoresis and Analysis 30 Load 30 Treatment 0, 1.25, 2.5 and 5 µm Etoposide Duplicates rows (24 wells/treatment) Inter run variability Single CometChip experiments on 3 different days Intra run variability Three Comet Chip experiment on same day

20 <20% Variability between treated wells Single CometChip experiments % DNA in Tail, n=24 well medians % DNA in Tail, n=24 well medians % DNA in Tail, n=24 well medians um Etoposide avg sd cv um Etoposide avg sd cv um Etoposide avg sd cv ~123 cells counted/well ~135 cells counted/well ~139 cells counted/well Three Comet Chip experiment on same day % DNA in Tail, n=24 well medians % DNA in Tail, n=24 well medians % DNA in Tail, n=24 well medians um Etoposide avg sd cv um Etoposide avg sd cv um Etoposide avg sd cv ~146 cells counted/well ~148 cells counted/well ~119 cells counted/well

21 <10% Variability between CometChips with treated samples inter run variability (n= 3 CometChips ) um Etoposide avg sd cv intra run variability (n=3 CometChips ) um Etoposide avg sd cv Low well to well and slide to slide CVs. Consistent with United States Pharmacopeia (USP) guidelines for 96 well assays (ELISA) Meets ASCLS specifications No overlapping comets. Minimal or no editing required results in Unbiased data analysis

22 CometChip Platform Provides Reproducible Data with Minimal Well to Well Variation % DNA in Tail (n=3) HepaRG: 4 hr MMS treatment µg/ml MMS Trt A Trt B HepaRG cells treated with increasing concentration of MMS. Two separate experiments performed by different operators on the same day. Each concentration performed in triplicate.

23 Cost Assumptions Traditional Comet Assay $50/hour fully burdened labor Full electrophoresis run (10 slides 20 wells) 200 comets per well Analyze 150 or more comets Five pictures/ well 12 hours per 20 wells (4000 comets) $698/run $ per well 17 cents per comet CometChip $50/hour fully burdened labor Full electrophoresis run 3 chips (288 wells) 400 comets per well Analyze 150 or more comets 1 picture / well 10.5 hours per 288 wells (115,200 comets) $1500/run $5.2 per well cents per comet

24 Traditional Comet: Cost per Run/Well Dollars spent on Traditional Comet Assays $12,000 $10,000 $8,000 $6,000 $4,000 $2,000 $0 Runs Wells Reagents & labor costs same as 3 CometChips $5,584 $4,886 $4,188 $3,490 $2,792 $2,094 $1,396 $698 $10,470 $9,772 $9,074 $8,376 $7,678 $6,980 $6,282 Buy the System Complete CometChip Run $1500 (288 wells) 9 Traditional Comet Assays $6282 (180 wells) Item Dollars Software 2995 CometChip System 3570 Subtotal % Discount CometChip Kits 650 Total 6555

25 CometChip Platform Developed and Manufactured under ISO 9001/2008 guidelines Only Integrated and Standardized System. CometChip Well Former and Electrophoresis Systems CometChip and Reagents Comet Analysis Software resulting in run to run consistency Low well to well and slide to slide CVs. Consistent with United States Pharmacopeia (USP) guidelines for 96 well assays (ELISA) No overlapping comets. Minimal or no editing required results in Unbiased data analysis

26 > 5000% increase throughput. CometChip Platform Develop and analyze over five hundred thousand comets per week Software can analyze 4X images requiring fewer fields or pictures to be examined Use existing microscope cameras or automated high through put cell analysis systems. (i.e Biotek or Celligo instrument s). Software validated using computer generated comet images with known comet tail parameters. Final software. Compatible JPEG or Tiff images Alkaline and neutral comet Compatible with CometChip and Standard Comet

27 CometChip for Human Skin Genotoxicity Testing Goal: To treat a 3D skin culture using a genotoxic agent and quantify DNA damage in basal keratinocytes. Issue: Can we preferentially select basal keratinocytes? Human epidermis during differentiation lose their nuclei creating a heterogeneous background of DNA damage in the comet assay Method: Can we use extracellular matrix proteins? Basal cells in skin stained with Anti-Integrin Beta 1 (green) Integrin Beta 1 binds to Collagen 1 in Extracellular Matrix

28 Collaborator: Dean Rosenthal, Georgetown University Supported by NIEHS R41ES Patent Pending DermaChip Identifies and Analyzes DNA Damage in Integrin Beta 1 Basal Cells of Skin EpiDerm TM Skin Model DermaChip (contains Collagen 1) Control (No treatment) Dissociate skin into single cells Stain cells with Anti-Integrin Beta 1 Load DermaChip Alkaline Comet Assay 100 nm H 2 O 2 Integrin Beta 1 mediates binding of Basal Cells to Collagen 1 in micropores Basal Cells co-stained with Anti-Integrin Beta 1 (orange) and Syber Gold (green)

29 Load Load Wash Washing removes cells not binding to Collagen 1 Jurkat Collagen 1 Chip Treated HaCat Collagen 1 Chip HaCat Collagen 1 Chip Jurkats suspension line lacking Beta 1 Integrin HaCat adherent line with Beta 1 Integrin Treated HaCat over trypsinized to remove surface receptors HaCat Chip only

30 Poster Sessions 1335: The Development and Validation of EpiComet-Chip, a Modified High- Throughput Comet. T. A. Townsend 1, M. C. Parrish 2, S. D. Shelton 1, B. P. Engelward 2, M. G. Manjanatha 1. 1 US FDA/NCTR, Jefferson, AR, United States. 2 Massachusetts Institute of Technology, Cambridge, MA, 2267: In Vitro Micronucleus and CometChip with Metabolically Competent HepaRG Cells. C. Swartz 1, J. Winters 1, K. Owens 1, C. Yauk 2, J. Buick 2, B. Engelward 3, L. Ngo 3, L. Recio 1. 1 Integrated Laboratory Systems, Research Triangle Park, NC, United States. 2 Health Canada, Ottawa, ON, Canada. 3 Massachusetts Institute of Technology, Cambridge, MA, United States.