Overview
This video demonstrates the methodology to screen for major cell death profiles in ionizing radiation (IR) induced cancer cell lines using DAPI staining assay. The DAPI-stained nuclei of target cells undergoing cell death exhibit distinct morphologies and can be easily identified by fluorescence microscopy.
Protocol
1. Preparation of Materials
- Autoclave coverslips
- Place coverslips in a glass beaker.
- Cover the glass beaker with foil.
- Autoclave at 121 °C at 15 psi for 20 min.
- Dry at 50 °C. Store at room temperature in a culture hood.
- Prepare the Fixation Solution: 3% paraformaldehyde + 2% sucrose in phosphate-buffered saline (PBS)
- To a 1,000 mL glass bottle, add 700 mL PBS and 30 g paraformaldehyde.
CAUTION: Paraformaldehyde is corrosive, wear appropriate gloves. - Dissolve paraformaldehyde completely by heating to 80 °C using a microwave.
CAUTION: Paraformaldehyde fumes are toxic, wear a mask. - Leave at room temperature overnight.
- Add 20 g sucrose.
- Add PBS to make the total volume 1 L.
- Aliquot in 50 mL tubes. Fixation Solution can be stored for 3 years at -20 °C or for 3 months at 4 °C.
- To a 1,000 mL glass bottle, add 700 mL PBS and 30 g paraformaldehyde.
2. Irradiation
CAUTION: Handle the X-ray irradiator carefully according to the manufacturer's instructions.
- Examine the cells under an inverted-phase microscope. Confirm that the cells are attached to the coverslip and alive.
- Irradiate the dish with 6 Gy X-rays (1.4 Gy/min, 300 KVP, 20 mA).
- 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
- Aspirate culture media from the dish.
- 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. - 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. - Place the dish in a square culture dish. Shake the square culture dish gently to distribute the Fixation Solution over the coverslip evenly.
- Incubate for 10 min at room temperature.
- Aspirate Fixation Solution from the dish.
- 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. - Aspirate PBS from the dish.
- 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
- 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. - Using a scalpel, remove the coverslip from the dish.
- Draw off the excess PBS on the coverslip by touching the edge of the coverslip with a paper towel.
- 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. - The samples can be stored for 1 year at -20 °C.
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Materials
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 |