Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy

Published: April 30, 2023

Abstract

Source: Lu, Y., et al. Evaluating In Vitro DNA Damage Using Comet Assay J. Vis. Exp. (2017)

In this video, we demonstrate an alkaline single-cell electrophoresis assay to quantify both single- and double-strand DNA breaks in doxorubicin-treated cells. Alkaline conditions disrupt the hydrogen bonds between DNA base pairs, resulting in the complete unwinding and separation of DNA strands. This allows differential migration of undamaged and damaged DNA fragments in agarose gel upon electrophoresis, revealing a comet-shaped structure of slowly migrating undamaged DNA forming the circular comet head, and rapidly migrating damaged DNA fragments forming the elongated comet tail.

Protocol

1. Prepare Reagents

  1. 1x PBS
    1. Dilute 100 mL 10x PBS with 900 mL dH2O and adjust the pH to 7.4 using a pH meter. Store at room temperature.
  2. Lysis solution (LS)
    1. Prepare 2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris base, and 200 mM NaOH in 900 mL dH2O; it commonly takes about 20 min to allow the mixture to fully dissolve. Adjust the pH to 10 using a pH meter. Add 1% sodium lauryl sarcosinate and 1% Triton X-100 and adjust the final volume to 1,000 mL. Cool to 4°C for at least 30 min before use.
  3. Alkaline electrophoresis solution (AES), pH >13
    1. Prepare 200 mM NaOH and 1 mM disodium EDTA in 800 mL dH2O. Adjust the pH and make sure that it is pH >13. Adjust the final volume to 1,000 mL. Make fresh before use and cool to 4°C for at least 30 min before use
  4. Staining solution
    1. 1. Add 1 μL 10,000x green fluorescent nucleic acid stain (e.g., SYBR Green) in 30 mL Tris-EDTA buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 7.4) and store at 4°C. Protect from light.
  5. 1% low melting agarose
    1. Melt 1% low melting point agarose (1 g in 100 mL dH2O) in a microwave. Swirl the agarose every 15-20 s to make sure that the agarose is completely molten. Place the agarose in 37°C water bath for at least 20 min before use.
  6. Pre-warm pipette tips
    1. Cut off the narrow ends of P200 pipette tips by 3 mm and warm at 37°C before pipetting agarose.

2. Prepare Comet Slides

  1. Slide coating
    1. Melt 1% agarose (1 g in 100 mL dH2O) in a microwave for 2 – 3 min or until the agarose is completely molten. Dip the glass microscope slides into the agarose and wipe one side of the slide using a lint-free wipe.
    2. Lay the slides on a flat surface to air-dry or heat at 50°C for faster drying; a transparent agarose film should be formed after drying. Place the coated slides in 37°C before use.
  2. Preparation of single cell suspensions
    1. Culture and treat the glioma cell
      1. Culture the U251 MG cells in DMEM-Ham F-12 medium supplemented with 10% FBS, 100 U/mL penicillin, and 10 μg/mL streptomycin at 37°C with 5% CO2.
      2. Digest the cells using 1 mL trypsin for 3 min and neutralize trypsin using DMEM-Ham F-12 medium with FBS. Collect in 15 mL tube, spin at 300 x g for 4 min, aspirate the medium, and suspend cells at 2 x 105 cell/mL in 1x PBS.
        NOTE: The cell sample should be prepared immediately before  starting the assay and all samples should be handled in a dark or dimmed environment to prevent DNA damage from light.
      3. Combine the cell suspension with 1% molten low melting point agarose (at 37°C) at a ratio 1:10 (v/v), mix gently by pipetting up and down, and immediately pipette 30 μL onto an agarose-coated slide. Use the side of the pipette tip to spread the agarose/cell mixture to ensure the formation of a thin layer.
      4. Place the slide flat at 4°C in the dark for 10 min. Increasing the   gelling time to 30 min improves adherence of samples in high humidity environments.
      5. Immerse the slide in 4°C LS in the dark for 1 h to overnight.
  3. Single Cell Electrophoresis
    1. For alkaline comet assay
      1. Gently remove slides from the LS, drain excess buffer, and gently immerse in AES for 1 h at 4°C to allow DNA unwinding. Keep the slides in the dark.
      2. Add pre-chilled AES in the electrophoresis slide tray, do not exceed 0.5 cm above the slides (this depends on the size of the electrophoresis units), place the slides inside and cover with a cap. Set the power supply voltage to 1 V/cm (the length between electrodes) and run for 30 min at 4°C.
      3. Drain excess electrophoresis solution from slide. Gently immerse slides twice in dH2O for 5 min each at room temperature.
      4. Gently immerse slides in 70% ethanol for 5 min at room temperature. Proceed to staining.
  4. Stain Comet Slides
    1. Dry slides at 37°C for 10 – 15 min in the dark.
    2. Place 50 – 100 μL green fluorescent nucleic acid staining solution onto each dried agarose and stain for 15 min at room temperature in the dark.
    3. Rinse the slides briefly in dH2O and dry completely at 37°C in the dark. Proceed to image acquisition and analysis.

Divulgazioni

The authors have nothing to disclose.

Materials

10x PBS(Ca++, Mg++ free)  TEKnova  P0196
NaCl  Sigma  S5886
EDTA  TEKnova  E0308
Trizma base  Sigma  T1503
NaOH  Sigma  72068
Sodium lauryl sarcosinate  Sigma  L7414
Triton X-100  Sigma  93443
SYBR Green  Invitrogen  S33102
Low melting point agarose  Invitrogen  16520
Agarose  Invitrogen  16500
95% ethanol  WARNER-GRAHAM  #64-17-5
Trypsin  GIBCO  25300-054
Glass tissue slides  ELECTRON MICROSCOPY SCIENCES  63422-11
Kimwipes  KIMberly-Clark
1.5 mL Microcentrifuge Tubes  DENVILLE
Pipette Tips  SHARP
Microwave  Avanti
Waterbath  PRECISION
Horizontal electrophoresis chamber  TREVIGEN  Cometassay ES II
Power supply  Bio-Rad
Incubator  Quincy Lab  Model 12-140E
Fluorescent microscope  Zeiss  LSM700
Micropipettor  Eppendorf

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Citazione di questo articolo
Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy. J. Vis. Exp. (Pending Publication), e21114, doi: (2023).

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