Experimental design

General Considerations

  1. Imaging media

    • Choose to minimise background fluorescence
      1. Avoid FBS and Phenol Red if possible
      2. Avoid Methylene Blue for IR imaging
      3. Some slide/well coatings are fluorescent
    • Will depend on metabolites required for cells, e.g.
      1. EBSS + 4mM Gln, 1mM NaPyruvate, 2mM CaCl2, 1 mM MgSO4 (Anca and Sean)
      2. HBSS (Chris) with additional HEPES if needed
      3. Invitrogen Fluorobrite (George)
  2. CO2 control required?
    • Required for some imaging media
    • Often possible to avoid CO2 if using appropriate buffered media (e.g. HEPES)
  3. Temperature control
    • Experiment at RT or 37°C?
    • Turn on incubator previous evening, heating stage several hours before
    • Use objective heater collar if using immersion fluid
    • Equilibrate dishes after media changes in incubator
    • Must allow temperature to equilibrate after positioning dish on microscope (10-15mins)
    • Monitor room temperature (USB temperature monitor available)
  4. Humidity
    • Consider evaporation during long term imaging
    • Humidify image chamber if possible
    • Seal plates if possible (but may lead to pH change if CO2 required)
    • Use layer of mineral oil on well
  5. Stimulation
    • Ideally use flow chamber
    • Control temperature of stimulation media
    • Consider diffusion effects if adding small volumes
    • Check for mechanical disturbance after adding stimulant
    • Always make control measurements with vehicle only, e.g. DMSO to check for flat baseline
    • Check for fluorescence from stimulants
  6. Imaging Substrate
    • Make sure you know the properties/thickness of coverslip/well bottom
    • Generally use No 1.5 coverslips
    • Clean coverslips with ethanol before use

Multi-well plate experiment design

Consider including the following control/calibration wells in your plate

  1. Suitable controls (will depend on sample), consider
    • Donor alone
    • Positive control (e.g. constitutively active protein)
    • Negative control (non-binding partner)
    • Donor with free acceptor
  2. Empty well with same imaging media
    • Treat the same as the other wells during plate setup during media change

  3. Untransfected cells

  4. Reference dye

Other issues to consider include

  • Plastic well plates are fluorescent in blue/green
    • Glass bottom plates preferable optically, but not compatible with some cells lines

  • Edge effects

  • Pipetting strategy

Choosing a reference dye

  • Reference dye should be a bright, mono-exponential fluorophore

  • Suggestions for common imaging channels
    • CFP channel: Green chromaslide
    • YFP channel: Green chromaslide
    • GFP channel: Green chromaslide
    • RFP channel: Rhodamine 6G

  • Chromaslides are good when monoexponential as no anisotropy artefacts

  • Reference dyes should be dissolved directly in water
    • Mixed solvents can lead to multi-exponential decay
  • Use low concentration, 10uM generally good
    • Avoid inner filter, re-absorbtion