Live Cell Imaging of EGF Binding to A431 Cells

Protocol – Live Cell Imaging of EGF Binding to A431 Cells

Reagents

• Tetramethylrhodamine-labeled recombinant EGF (ThermoScientific)
• Imaging media: 117 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4, 20 mM HEPES, Glucose, BSA
  • NaCl: 6.83 hg/L
  • KCl: 0.395 g/L
  • HEPES: 4.77 g/L
  • NaH2PO4-2H2O: 0.195 g/L
  • Glucose: 9g/L
  • BSA: 100mg/L (add after autoclave)
  • Adjust PH to 7.2 after autoclave
• A431 cell (ATCC)

Equipment

• Interchangeable coverglass dish (ICD) with lid (Bioptechs Inc)
• 30 mm round coverglass (Bioptechs Inc)
• Stable-Z temperature-controlled microscopy stage (Bioptech Inc)
• Objective heater (Bioptech Inc)
• HILO fluorescence microscope (home-build using Olympus IX frame)
• Standard fluorescence filter set for TMR (Semrock Inc)
• Plasma cleaner (Harrick Plasma)

Procedure

1. Culture A431 cells according to the ATCC protocol (DMEM medium supplemented with 10% FBS).
2. Two days before imaging experiment: clean/sterilize 30-mm coverglass with an O₂ plasma cleaner (medium power, 3min).
3. Seed A431 cells on coverglass at low density (~10,000 cell per coverglass) according to the standard sub-culture protocol. Culture normally for 24 hours.
4. One day before the imaging experiment: replace culture medium with serum-free DMEM medium. Culture overnight.
5. On the day of imaging experiment: Set the temperatures of the microscopy stage and objective heater to 37 °C. 
6. Prepare imaging media and TMR-EGF-containing imaging media to 2x of the desired concentration, e.g., for final concentration of 25 ng/ml EGF, prepare imaging media with 50 ng/ml TMR-EGF. Pre-warm all imaging media to 37 °C.
7. Assemble imaging chamber using ICD and the glass coverglass. Add 1ml pre-warmed EGF-free imaging medium before covering with lid. Install the imaging chamber on the microscope to allow the temperature to re-equilibrate.
8. Using cell auto-fluorescence as a cue, adjust HILO excitation angle to be minimal yet still allow whole-cell illumination. 
9. Reduce field-aperture to be as small as possible, but allowing single-cell imaging.
10. Engage autofocus.
11. Start time-lapse imaging at 0.1-0.2 Hz imaging speed. While recording images, using a syringe, add 1-ml pre-warmed EGF-containing imaging medium via the perfusion port of the ICD lid into the imaging chamber.
12. Fluorescence signal at the cell should quickly rise and typically reach the saturation level within minutes. A typical result is shown below.

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