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

www.trevigen.com | 301-216-2800 | info@trevigen.com8405 Helgerman Court, Gaithersburg MD 20817

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

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 1988.

• 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.

Principles of the Comet Assay

Cells placed in LM Agarose

Immobilize cells on CometSlide™

Treat with Lysissolution

Alkali Treatment

Alkaline Electrophoresis and stain

Large Fragments

Small Fragments

Typical Comet Data

The CometChip® Platform

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

CometChip®

Trevigen’s CometChip® Platform: Reagents, Apparatus and SoftwareCometChip® Well Former CometChip® Electrophoresis Unit with Power Supply

CometChip® Reagents Comet Analysis Software

CometChip® provides high density data with minimal overlaps compared to older comet formats

Older Comet Format CometChip®

Trevigen® Comet Analysis System(Analyze >500,000 comets per week)

SCAN EDITANALYZE

“Wizard Driven Software”

Free Demonstration at Booth 1728

Directions from Wizard

Study Name

Drag and Drop Images

Automatic Comet finding and Scoring

Edit based on profile

Review from Image

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 < 10-15 minutes. • Minimal editing due to no over lapping comets.

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.

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.)

Excellent Correlation Between Expected and Program Generated Analysis of Simulated Comets

R² = 0.9946

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R² = 0.9904

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% DNA in Tail

R² = 0.9849

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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.

Trevigen Analysis System Provide Comparable Resultsfor 4X and 10X Images in Standard Neutral Comet Assays

R² = 0.9998

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10X Image

% DNA in Tail

R² = 0.99130

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Tail Length

R² = 0.9974

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Moment

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.

R² = 0.9888

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R² = 0.9841

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Loats automated system (10X)

Trevigen Analysis System Provide Comparable Results for 4X and 10X Images in the Standard Alkaline Comet Assay

4X vs 10X tiff images: 1:1 correlation between Trevigen software and Loats automated system

% DNA in Tail Comparisons

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% Variability between treated wells

uM Etoposide avg sd cv uM Etoposide avg sd cv uM Etoposide avg sd cv0 4.64 1.59 34.23 0 4.41 1.02 23.18 0 6.05 1.55 25.70

1.25 20.10 3.50 17.40 1.25 21.94 3.46 15.77 1.25 22.38 4.12 18.402.5 34.30 3.80 11.09 2.5 38.70 2.75 7.12 2.5 38.99 2.84 7.295 53.59 4.19 7.82 5 57.57 3.90 6.77 5 61.94 6.49 10.48

~123 cells counted/well ~135 cells counted/well ~139 cells counted/well

% 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 cv0 4.78 1.02 21.24 0 4.86 0.84 17.33 0 6.64 1.67 25.12

1.25 19.36 2.12 10.95 1.25 18.11 1.51 8.35 1.25 21.87 2.35 10.732.5 32.74 2.32 7.09 2.5 31.00 2.51 8.10 2.5 34.93 2.74 7.845 61.71 6.47 10.48 5 61.94 5.48 8.85 5 55.30 7.49 13.54

~146 cells counted/well ~148 cells counted/well ~119 cells counted/well

% DNA in Tail, n=24 well medians % DNA in Tail, n=24 well medians % DNA in Tail, n=24 well medians

Single CometChip® experiments

Three Comet Chip® experiment on same day

1 2 3

<10% Variability between CometChips® with treated samples

inter run variability (n= 3 CometChips®)uM Etoposide avg sd cv

0 5.03 0.89 17.611.25 21.47 1.21 5.642.5 37.33 2.63 7.045 57.70 4.18 7.24

intra run variability (n=3 CometChips®)uM Etoposide avg sd cv

0 5.43 1.05 19.331.25 19.78 1.91 9.682.5 32.89 1.97 5.995 59.65 3.77 6.32

• 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

CometChip® Platform Provides Reproducible Data with Minimal Well to Well Variation

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HepaRG: 4 hr MMS treatment

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.

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• $ 34.90 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• 1.3-2.0 cents per comet

Traditional Comet: Cost per Run/Well

$698 $1,396

$2,094 $2,792

$3,490 $4,188

$4,886 $5,584

$6,282 $6,980

$7,678 $8,376

$9,074 $9,772

$10,470

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$2,000

$4,000

$6,000

$8,000

$10,000

$12,000

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Buy the System

Reagents & labor costs same as 3 CometChips®

Runs

Wells

Item Dollars

Software 2995

CometChip® System 3570

Subtotal 6565

- 10% Discount 5905

2 CometChip® Kits 650

Total 6555

1 Complete CometChip Run---------$1500 (288 wells)9 Traditional Comet Assays----------$6282 (180 wells)

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

CometChip® Platform• > 5000% increase throughput.

– 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 Biotekor 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

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?

Integrin Beta 1 binds to Collagen 1 in Extracellular Matrix

Basal cells in skin stained with Anti-Integrin Beta 1 (green)

DermaChip™ Identifies and Analyzes DNA Damage in Integrin Beta 1 Basal Cells of Skin

EpiDermTM Skin Model

Dissociate skin into single cells

Alkaline Comet Assay

Control (No treatment)

100 nM H2O2

DermaChip™(contains Collagen 1)

Collaborator: Dean Rosenthal, Georgetown UniversitySupported by NIEHS R41ES026908Patent Pending

Basal Cells co-stained withAnti-Integrin Beta 1 (orange)

and Syber Gold (green)

Collaborator: Dean Rosenthal, Georgetown UniversitySupported by NIEHS R41ES026908Patent Pending

Load DermaChip™

Stain cells with Anti-Integrin Beta 1

Integrin Beta 1 mediates binding of Basal Cells to Collagen 1 in micropores

Washing removes cells not binding to Collagen 1

Load

JurkatCollagen 1

Chip

HaCatCollagen 1

Chip

HaCatChip only

Treated HaCatCollagen 1

Chip

Load Wash

Jurkats – suspension line lacking Beta 1 IntegrinHaCat – adherent line with Beta 1 IntegrinTreated HaCat – over trypsinized to remove surface receptors

Poster Sessions1335: The Development and Validation of EpiComet-Chip, a Modified High-Throughput Comet. T. A. Townsend1, M. C. Parrish2, S. D. Shelton1, B. P. Engelward2, M. G. Manjanatha1. 1US FDA/NCTR, Jefferson, AR, United States. 2Massachusetts Institute of Technology, Cambridge, MA,

2267: In Vitro Micronucleus and CometChip® with Metabolically Competent HepaRG™ Cells. C. Swartz1, J. Winters1, K. Owens1, C. Yauk2, J. Buick2, B. Engelward3, L. Ngo3, L. Recio1. 1Integrated Laboratory Systems, Research Triangle Park, NC, United States. 2Health Canada, Ottawa, ON, Canada. 3Massachusetts Institute of Technology, Cambridge, MA, United States.