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

Protokoll

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.

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Materialien

Name	Company	Catalog Number	Comments
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