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Lab 7

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22 views14 pages

Lab 7

Uploaded by

queensneverdie2015
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Pharmacology and Toxicology

department

Single Cell Microgel Electrophoresis


technique

Toxicology Lab 7
Winter 2021

1
ILOS
• Recognize the principles of Comet assays technique.
• Studying the principle and procedures of cell culture preparation. Watching live
video for cell culture preparation.
• Staining, counting and calculating the number of viable cells using Neubar
Hemocytometer, under light microscope.
• Identify & describe the preparation of microgel step (slide treatment step).
• Study the exact function the buffers used in the preparation of microgel step.
• Visualizing and evaluating the obtained comet and healthy cells under the
fluorescent microscope.
• Communicate clearly in the laboratory sessions by verbal means through oral
questions.
• Write a lab report for the obtained results.

2
Lab content

• Aim of experiment
• Procedures steps (Step II and Step III)
• Step II: Slide treatment steps
• Step III: Evaluating DNA damage due to
Bleomycin using fluorescent microscope.

3
Aim of the experiment

Detecting the extent of DNA damage due


to application of an approved genotoxic
drug Bleomycin to HEK293 cells using
alkaline single cell gel electrophoresis
assay

4
Procedures Steps

III)
II)
I) Evaluation
Steps of of DNA
Cell culture comet assay
preparation damage
experiment using
Slide treatment
steps Fluorescent
microscope

5
Steps of comet assay experiment
Slide treatment steps

1. Fixation Step: 1a& 1b


2. Lysis Step
3. Electrophoresis step
4. Staining step

6
Steps of comet assay experiment
Slide treatment steps
Reagents and tools needed

1. a. Eppendorf, bleomycin& Low melting


point agar (LMP)
1.b.Frosted end slide& normal melting point
agar(NMP)
2. Couplin jar& lysis buffer
3. Dark electrophoresis chamber& alkaline
buffer
4. Rack& Dye: Ethidium bromide

7
Steps of comet assay experiment
Slide treatment steps
Procedure

1.a. Fixation step:


 In an eppindorff:
• 1 x 106 cells incubated with bleomycin; then suspended in low melting point agar (LMP) Agar.
 N.B: LMP is used not normal melting point agar (NMP) as cells die at melting point of NMP
agar
1.b. Fixation step:
 On frosted slides
• Cells are fixed on frosted slides previously coated with NMP Agar.
N.B: In Fixation step: Slides are kept in the dark: To avoid UV–light induced DNA damage; and
kept at 4˚C: to avoid activation of DNA repair mechanisms.

1a 1b

Microgel

8
Steps of comet assay experiment
Slide treatment steps
Procedure continued

2.Lysis step:
The slides are dipped in lysis buffer (pH 10) in a Couplin Jar and incubated at 4˚C for 1 hour.
 The slides are washed with neutralizing buffer (pH 7) to remove traces of the lysis buffer.
3. Electrophoresis step:
• The slides are submerged in Electrophoresis Alkaline Buffer (pH 13) and Electrophoresis is conducted
for 1 hour then neutralize with neutralizing buffer (pH 7).
4. Staining step:
• Stain the slides with Ethidium Bromide, incubate in the dark for 5 minutes; get rid of excess of
Ethidium bromide by decantation and then dry the slide.

2 3 4

9
Experimental procedure
slide Treatment Steps
Functions of Buffer

Lysis Buffer • Removes nuclear membranes and


histones.
pH 10

Electrophoresis • The buffer acts as a conducting


solution during electrophoresis.
alkaline buffer • Alkaline condition unwind the
DNA double stranded helix to
pH 13 detect single strand breaks.

10
III. Imaging under the fluorescent microscope
(Evaluation of the DNA)

Head + Tail= Comet

11
Evaluating comets by Image
Analysis Software

• It is the distance of DNA migration from the nucleus.


Tail length
• Not very useful as it doesn't reflect the actual damage levels.

• It is the relative fluorescence intensity of head and tail (expressed as percentage of


DNA in tail)
• The most useful parameter
Tail intensity • Has linear relationship to break frequency.

• It is the product of tail length and intensity


Tail Moment

12
Evaluating comets by Image Analysis Software
Criteria for comet selection

1. Comets must be selected without bias

2. Edges and areas around air bubbles should be avoided

3. No overlapping comets

13
14

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