Comet Assay

CometAssay
Society of Toxicology Exhibitor Hosted Session Trevigen March 18, 2008 Sandra Woodgate, PhD Thomas Uveges, PhD
ams biotechnology (europe) ltd info@amsbio.com www.amsbio.com (UK) +44(0)1235 828200 (CH) +41(0)91 604 55 22 (DE) +49(0)69 779099
Background
The Comet Assay is a single cell gel electrophoresis assay for evaluating DNA damage in cells
Based on denatured, cleaved DNA fragments migrating out of the cell when a current is applied whereas undamaged DNA migrates slower
Applications
Study cellular response to DNA damage Screening assay for inhibitors to DNA repair (cancer therapy) Testing chemicals for toxicity
Assay Overview
1.
1. 2. 3.
Cells mixed with low melting agarose at 37C (LM Agarose) Immobilize cells on CometSlide Treat cells with Lysis Solution (removes membranes and histones from the DNA) Samples treated with alkali (unwinds and denatures DNA) Alkaline electrophoresis performed (reveals DNA breaks) Samples stained with intercalating dye and visualized by epifluorescence microscopy.
6. 3. & 4.
2.
5.
4. 5. 6.
Sybr Green I
DNA migration out of nucleoid
Comet Definitions
Percent DNA in the Tail
The integrated tail intensity x 100 divided by the total integrated cell intensity for a normalized measure of the percent of total cell DNA found in the tail
Tail Moment
The product of distance and normalized intensity integrated over the tail length, (Lx % DNAx) a damage measure combining the amount of DNA in the tail with the distance of migration (severity of damage)
Alkaline Electrophoresis Conditions

Standard Procedures
Singh et al. 1988 Olive and Banath, 2006 http://cometassay.com
Fresh Alkaline Buffer and large horizontal tank

Cold 1 mM EDTA and 300 mM NaOH, pH>13 Add buffer so barely covering slides Electrophoresis for 20-40 min
0.7 V/cm or 1 V/cm at 300 mA
Electrophoresis
Variables: Alkaline Electrophoresis

Electrode Distance and Voltage
1 volt/cm (300 mA) is recommended. Amperage problem with some tanks.
Buffer Height
More results in less electrophoresis.
Buffer Temperature
Alkali solutions are not buffered and great variations occur, recirculation and/or cold electrophoresis works best.
Electrophoresis Time
Longer tails with longer electrophoresis time.
Variables: Alkaline Electrophoresis

Buffer Effects on Amperage
Different comet shapes and tail lengths by changing pH of electrophoresis solutions High buffer concentrations affect amperage
Position equal distance between electrodes

To minimize well to well variation
Slide Orientation
Movement during electrophoresis affects measurements
Control Cells
Control Cells to standardize and compare electrophoresis methods between users and laboratories.
Four unsynchronized suspension cell preparations with incremental increases of DNA damage Designed to be used when performing alkaline electrophoresis Designed for long term storage
Control Cells: % DNA in Tail

90 70 % DNA in Tail 50 30 10 -10 CCO CC1 CC2 CC3 Control Cells
% DNA by Etoposide CCO CC1 CC2 CC3
Four statistically distinct populations
n 50 50 50 50
Mean 5.757 28.374 39.736 56.800
SD 7.7270 14.0080 21.8164 23.5893
SE 1.0928 1.9810 3.0853 3.3360
75% CI of Mean 4.485 to 7.029 26.068 to 30.680 36.144 to 43.328 52.916 to 60.683
Median 1.640 28.990 37.050 51.905
IQR 8.925 20.313 32.183 40.240
75% CI of Median 1.290 to 2.230 25.180 to 31.840 27.790 to 44.630 45.460 to 64.390
Electrophoresis Tanks
In-house
Bio-Rad DNA Sub-Cell
older model
Developed for Comet

Cleaver Scientific 28 cm x 25 cm (L)
16 cm x 30 cm (L)
Optimize Running Conditions

Bio-Rad Running Conditions
~1L Alk Buffer pH>13 20 at 1V/cm (300 mA)
Cleaver 150 mM*

% DNA (S.D.) 9.46 (1.47) 31.2 (2.45) 43.4 (2.41) 66.2 (0.9)
300 mM
% DNA (S.D.) 6.7 (0.86) 32.4 (2.03) 44.08 (1.63) 60.6 (3.62)
Control Cells (5 replicates)

CO C1 C2 C3
* Not possible to always adjust to 300 mA by buffer height

0.4 cm with 300 mM NaOH is > 500 mA
40 at 0.7 V/cm (300 mA) using 300 mM NaOH 20 at 1 V/cm (300 mA) using 150 mM NaOH
Alkaline Buffer Concentration

pH 10 11 12 13 14 [OH-] (M) 10-4 10-3 10 mM 100 mM 1M
300 mM NaOH, pH >13

typically recommended to maintain strand denaturation Some users unable to run at 1V/cm due to high amperage
pH 13 is achieved at concentrations > 100 mM NaOH

Vary Slide Position on Platform

Anode C3
+
Cathode C3
Differences in Comet shape

Position dependent Faster migration near cathode (length)
# Counted C2 - A C C3 - A C
BioRad Unit
TM 9.04 14.8 15.49 23.03
% DNA 43.36 51.27 59.38 67.69
Length 38.4 54.2 46.75 61.60
111 111 84 94
30 cm electrode distance/300 mM NaOH
Designed Prototype Tank

Amperage and Temperature [NaOH] pH>13 Electrode distance and Buffer height Slide Position
Platform
1 2 3 4 5 6 Electrode positions
7 8 9 10 11 12
Electrode positions
Amperage
Electrode Distance (0.4 cm buffer)
500
(constant 1V/cm)
Buffer Height (16 cm distance)

500 (constant 1V/cm)
mAmps
400 300 200 100 11 13 15 17 19 21
150 mM NaOH - 4 C 300 mM NaOH - 4 C 300 mM NaOH - 24 C
mAmps
150 mM NaOH - 24 C
400 300 200 100 0.4 0.6 0.8 1 Buffer Height (cm)
V=IR
Electrode Distance (cm)
At constant volts, Amperage increases with

Electrode Distance Temperature [NaOH] Buffer Height
300 mM NaOH problematic for 400 mA powerpacks

Electrophoresis at 4C is needed keep amperage <400 mA)
[NaOH] pH >13
150 mM
C1
Comet Length vs NaOH
80
200 mM
C1 C3
300 mM
C1 C3
C3
Length
60 40 20 0 150 200 250 300
C1
C3
mM NaOH
30 min/21 cm 1V/cm, constant buffer height
Length increases with lower [NaOH] Better sensitivity w/ 150-200 mM NaOH

Electrode Distance Buffer Height

Length/Buffer Height vs Electrode Distance
C2 Length Buffer Height (micron)
Adjusting to 300 mA using buffer height reduces Comet length as electrode distances are decreased
Buffer height >0.5 cm reduces length Electrode distance < 18 cm reduces length
CO C1 C2 C3 CO C1 C2 C3 CO C1 C2 C3
120 100 80 60 40 20 0
13 15 17 19 21 Buffer Height C2 Length
Electrode Distance (cm)
Electrophoresis - 1V/cm (300 mA), 150 mM NaOH

CO C1 C2 C3 CO C1 C2 C3
Elect Dist:
13 cm
16 cm
18 cm
21 cm
Control Buffer Height - Overlay

0.4 cm buffer height
Low Buffer
70 60 50 40 30 20 10 0 1 2 3
70 60 50 40 30 20 10 0 1 2 3
1 cm buffer height
High Buffer
70 60 50 40 30 20 10 0 1
Overlay
High buffer- overlay
% DNA
% DNA
CO
C1
C2
C3
CO
C1
C2
C3
% DNA
C0
C1
C2
C2
2 3
Well Position
Well Position
Well Position
Piece of plastic placed over slides Reduces variation due to buffer height

Vary Slide Position on Platform

CO C1 C2 C3
Stacking Slides increases Variation

~50% Voltage Drop across slide platform
Top
# Counted
CO T B
CO C1 C2 C3
TM 0.54 0.02 3.07 0.46 4.84 0.68 7.48 0.39
% DNA 3.92 0.96 21.48 5.12 29.23 5.96 40.35 4.73
Length 3.7 1.2 18.8 4.8 23 5.4 29.2 4
208 321 259 293 135 139 139 111
Bottom
C1 T B C2 T B C3 T B
Model Tank
21 cm in length Run at 1V/cm, ~300 mA for 30-40 minutes
Use with most laboratory power supplies
No well to well variation

Overlay to maintain 0.4-0.5 cm buffer height 1L of buffer, 150-300 mM NaOH pH >13
Platform accommodates 1 row of slides

Maintain slide position Use 2, 3, 20 and 96 well slide formats Easy removal and placement of slides
Maintain buffer temperature ~4C

Ceramic plate and cooling chamber
Exploded Diagram
Interlocking Safety Lid
Overlay
2/20 Well Tray Ceramic
Water Chamber Ports
Samples
2/20 well Tray
Ten 2-well slides Five 20-well slides
96 well Tray
3 slides - 288 samples
96 Well Tray
Alkaline pH >13
% DNA in Tail 200 mM NaOH
220 mA for 30
% DNA in Tail 300 mM NaOH

380 mA for 40
CO C1 C2 C3
7.21 24.57 40.39 56.63
7.29 27.64 40.71 55.96
Good sensitivity in 30 w/ 200 mM NaOH (< 300 mA) Or 40 w/ 300 mM NaOH (>300 mA) Achieved Specifications for Control Cells
1 V/cm using 2 well slides
96 well slide
Minimal well to well variation with Slide Overlay
70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12
% DNA in Tail
CO
C1
C2
C3
Well Position (cathode -> anode)
60 50
Length
40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12
CO
C1
C2
C3
Well Position
Buffer Height Maintained with Slide Overlay

% DNA
+ Overlay - Overlay
Length
+ Overlay - Overlay 12.38 31 39.08 48.17 4.67 10.5 19.08 24.7 6.2
CO C1 C2 C3
11.76 29.76 43.47 59.94
13.93 26.09 35.25
Buffer height increases without slide overlay

% DNA in Tail Decreases Length Decreases
Comet Parameters Slide Format

CO 2 well 20 well 96 well 2 well 20 well 96 well 2 well 20 well 96 well 2 well 20 well 96 well TM % DNA Length 1.42 7.21 15 1.64 7.10 17 1.62 11.76 12 4.62 24.57 36 6.27 28.69 39 5.11 29.76 31 10.45 40.39 50 10.96 42.99 47 9.07 43.47 39 16.39 56.63 55 15.98 55.71 54 16.02 59.94 48
30 'electrophoresis for 2/20 well slides 40' electrophoresis for 96 well slides
C1
C2
C3
1V/cm, 200 mM > pH 13
Modified Comet Assay DNA Damage

1. Cells Mixed with LM Agarose
Spread 40 l 1 hr at 4C 3x 10 at RT 45 at 37 Hydrophobic coverslip
Type of Damage
UV Damage Oxidative Damage AP sites
2. Treat cells with Lysis Solution 3. Equilibrate with Enzyme Buffer 4. Treat with Enzyme
DNA Repair Enzymes

Lesion Specific Bacterial enzymes
5. 6. 7.
Treat with Alkali unwind Alkaline electrophoresis Samples stained and visualized
Repair Enzymes
FPG
glycosylase for oxidized purines with associated AP lyase 8-oxoGua, FaPyGua, FaPyAde
other ring-opened purines
ENDO III
glycosylase for oxidized pyrimidines with associated AP lyase
including thymine glycol and uracil glycol
hOGG1
glycosylase for oxidized purines with cleavage of phophodiester bond through Schiff base chemistry 8-oxoGua, FaPyGua
Oxidative Damage with KBrO3

% DNA in Tail
No Enzyme Healthy Cells Treated Cells 4.3 5.0 FPG 21.3 * 33.4 * hOGG1 7.7 13.0* ENDOIII 8.2 6.0
FPG showed a dramatic increase in breaks for Healthy cells compared to hOGG1 and ENDOIII FPG and hOGG1 showed a similar increase in breaks with Treated Cells compared to Healthy cells but not for ENDOIII. hOGG1 more specific for oxidative damage with KBrO3
Mutagenesis 2006
* significant
Monitor DNA Recovery

U T 1hr 2 hr
Untreated H202 Treated 1 hr Recovery 2 hr Recovery TM % DNA Length 1.0 3.4 22 38.6 7.4 2.7 87.3 25.5 9.4 71 55.3 28
H2O2 treatment - high level of damage 2 hr recovery - reduction in all comet parameters
Alkaline Comet
Electrophoresis Buffers
Alkaline Buffer
Droplet comet shape
Neutral Buffer (TBE)

Elongated comet shape
Greater sensitivity
ss- and ds-breaks alkali-labile sites (AP sites)
Reduced sensitivity
due to DNA renaturation Detection of ds-breaks
DNA Repair of Double Strand Breaks

DNA DAMAGE (Etoposide) Activation of DNA-PK pathway for NHEJ repair
Treat with PI3Kinase inhibitor (Wortmannin)
Recovery from DNA damage
Inhibit DNA-PK pathway
Unable to repair DS breaks by DNA PK mediated NHEJ Cell unable to pass cell cycle checkpoints If level of damage high enough and/or unable to repair damage Cell death occurs
Neutral Comet
# Untreated Damaged Recovery Inhibitor
95 138 74 114 88 78 95 64
TM %DNA
0.98 0.95 2.23 2.19 0.63 0.34 1.94 1.93 7.66 8.34 14.15 15.57 6.94 4.63 14.09 11.03
Length
8 10 19 19 10 6 19 16
Double-strand break repair in 1 hr PI3 kinase inhibitor Wortmannin inhibits ds repair Damage levels remain same in presence of inhibitor
Summary
Control Alkaline Electrophoresis
Optimized Electrophoresis System CometAssay Control Cells To monitor DNA Damage and Repair
Patent Pending
CometAssay Kits
Lysis Solution LMAgarose CometSlide 2, 20 or 96 well Staining Reagent SYBR Green Silver Staining Reagents
FLAREFormats
CometAssayKit Repair Enzyme Oxidative and Chemical Damage Fpg, hOGG1 UNGase, Endonuclease III Endonuclease IV UV Damage T4-PDG, cv-PDG UVDE

Comet Assay

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Comet Assay

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CometAssay

Alkaline Electrophoresis Conditions

Fresh Alkaline Buffer and large horizontal tank

Variables: Alkaline Electrophoresis

Variables: Alkaline Electrophoresis

Position equal distance between electrodes

Control Cells: % DNA in Tail

% DNA by Etoposide CCO CC1 CC2 CC3

Four statistically distinct populations

Mean 5.757 28.374 39.736 56.800

SD 7.7270 14.0080 21.8164 23.5893

SE 1.0928 1.9810 3.0853 3.3360

Median 1.640 28.990 37.050 51.905

IQR 8.925 20.313 32.183 40.240

Developed for Comet

Optimize Running Conditions

Cleaver 150 mM*

Control Cells (5 replicates)

* Not possible to always adjust to 300 mA by buffer height

Alkaline Buffer Concentration

300 mM NaOH, pH >13

pH 13 is achieved at concentrations > 100 mM NaOH

Vary Slide Position on Platform

Differences in Comet shape

TM 9.04 14.8 15.49 23.03

% DNA 43.36 51.27 59.38 67.69

Length 38.4 54.2 46.75 61.60

30 cm electrode distance/300 mM NaOH

Designed Prototype Tank

Buffer Height (16 cm distance)

400 300 200 100 11 13 15 17 19 21

150 mM NaOH - 4 C 300 mM NaOH - 4 C 300 mM NaOH - 24 C

Electrode Distance (cm)

At constant volts, Amperage increases with

300 mM NaOH problematic for 400 mA powerpacks

60 40 20 0 150 200 250 300

30 min/21 cm 1V/cm, constant buffer height

Length increases with lower [NaOH] Better sensitivity w/ 150-200 mM NaOH

Electrode Distance Buffer Height

Electrode Distance (cm)

Electrophoresis - 1V/cm (300 mA), 150 mM NaOH

Control Buffer Height - Overlay

21 cm electrode distance/300 mM NaOH

Vary Slide Position on Platform

Stacking Slides increases Variation

TM 0.54 0.02 3.07 0.46 4.84 0.68 7.48 0.39

% DNA 3.92 0.96 21.48 5.12 29.23 5.96 40.35 4.73

Length 3.7 1.2 18.8 4.8 23 5.4 29.2 4

208 321 259 293 135 139 139 111

No well to well variation

Platform accommodates 1 row of slides

Maintain buffer temperature ~4C

2/20 Well Tray Ceramic

Water Chamber Ports

% DNA in Tail 300 mM NaOH

7.21 24.57 40.39 56.63

7.29 27.64 40.71 55.96

Well Position (cathode -> anode)

Buffer Height Maintained with Slide Overlay

11.76 29.76 43.47 59.94

13.93 26.09 35.25

Buffer height increases without slide overlay