Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines

Protocolo

1. Preparation of Materials

1. Autoclave coverslips
   1. Place coverslips in a glass beaker.
   2. Cover the glass beaker with foil.
   3. Autoclave at 121 °C at 15 psi for 20 min.
   4. Dry at 50 °C. Store at room temperature in a culture hood.
2. Prepare the Fixation Solution: 3% paraformaldehyde + 2% sucrose in phosphate-buffered saline (PBS)
   1. To a 1,000 mL glass bottle, add 700 mL PBS and 30 g paraformaldehyde.
      CAUTION: Paraformaldehyde is corrosive, wear appropriate gloves.
   2. Dissolve paraformaldehyde completely by heating to 80 °C using a microwave.
      CAUTION: Paraformaldehyde fumes are toxic, wear a mask.
   3. Leave at room temperature overnight.
   4. Add 20 g sucrose.
   5. Add PBS to make the total volume 1 L.
   6. Aliquot in 50 mL tubes. Fixation Solution can be stored for 3 years at -20 °C or for 3 months at 4 °C.

2. Irradiation

CAUTION: Handle the X-ray irradiator carefully according to the manufacturer's instructions.

1. Examine the cells under an inverted-phase microscope. Confirm that the cells are attached to the coverslip and alive.
2. Irradiate the dish with 6 Gy X-rays (1.4 Gy/min, 300 KVP, 20 mA).
3. Incubate for 72 h at 37 °C in an atmosphere containing 5% CO₂.
   NOTE: Incubation time can be modified according to the experimental design

3. Fixation

1. Aspirate culture media from the dish.
2. Using scissors, cut the tip of a 1,000 μL micropipette tip 5 mm from the end.
   NOTE: The cut tip helps to smoothly apply the Fixation Solution.
3. Using the cut tip, add 1 mL Fixation Solution (prepared in step 1.2) to the dish from the sidewall of the dish.
   NOTE: This step is time-sensitive and should therefore be performed without changing micropipette tips when handling multiple samples. Application of Fixation Solution directly to the bottom of the dish can damage the cells.
4. Place the dish in a square culture dish. Shake the square culture dish gently to distribute the Fixation Solution over the coverslip evenly.
5. Incubate for 10 min at room temperature.
6. Aspirate Fixation Solution from the dish.
7. Add 2 mL of PBS to the dish from the sidewall of the dish.
   NOTE: Application of PBS directly to the bottom of the dish can damage the cells.
8. Aspirate PBS from the dish.
9. Repeat steps 3.7 and 3.8 twice.
   NOTE: The dish can be stored for 1 week at 4 °C with the coverslips bathed in 2 mL PBS.

4. DAPI Staining

1. To a glass slide, apply 5 μL 1 μg/mL DAPI staining reagent.
   NOTE: The DAPI staining reagent is stable for 6 months when stored protected from light at or below -20 °C.
2. Using a scalpel, remove the coverslip from the dish.
3. Draw off the excess PBS on the coverslip by touching the edge of the coverslip with a paper towel.
4. Mount the coverslip upside-down onto the drop of DAPI staining reagent on the glass slide so that the cells are exposed to DAPI staining reagent.
   NOTE: This step is time-sensitive. Drying of the DAPI staining reagent can lead to suboptimal staining.
5. The samples can be stored for 1 year at -20 °C.

Materiales

Name	Company	Catalog Number	Comments
Cover slip	Matsunami	C013001
Paraformaldehyde	Sigma-Aldrich	P6148-1KG
Sucrose	Sigma-Aldrich	84097-250G
Phosphate-buffered saline	Cosmo Bio	16232000
Scalpel	Kai Medical	No.20, 520-A
35 mm cell culture dish	Falcon	353001
inverted phase microscope	Shimazu	114-909
X-ray irradiator	Faxitron Bioptics	Faxitron RX-650
square culture dish	Corning	431110
slide glass	Matsunami	Pro-11
DAPI staining reagent	Cell Signaling	8961S
Fluorescence microscope	Nikon	ECLIPSE Ni
fluorescence microscope DAPI filter	Nikon	U-FUW, BP340-390, BA420IF, DM410
fluorescence microscope lens (20×)	Nikon	Plan Apo λ 20X/0.75
fluorescence microscope lens (60×, oil)	Nikon	Plan Apo λ 60X/1.40 oil
oil	Olympus	N5218600
CCD camera	Nikon	DS-Qi2
digital image acquisition software	Nikon	NIS-Elements Document
lens cleaner	Olympus	OT0552
chloroform	Wako	035-02616