Live cell microscopy

Preparation of culture dishes

• In 3.5 cm culture dishes (Sarstedt 83.1800 35x10 mm) a hole is drilled with 14 mm in diameter (will be done by the uni garage)
• Before sticking the coverslips (Menzel 24 mm, Stärke 1,5; CB00240RAC20MNZ#0) to the dishes spray them with 70% ethanol and wipe with a KimWipe tissue
• The coverslips are pretreated as following:
  • Wash with a mixture of 50% v/v Aceton and 50% v/v EtOH
  • Wash with 1xPBS
  • Wash with MilliQ water
  • Dry and sterilize at 120°C over night
• Stick the coverslip with Fixogum (Marabu) to the culture dishes
• Dry for two days in the dark

Coating of culture dishes and seeding of cells

• Culture dishes are sterilized for 5 min at 850 W in a microwave (add a bottle of dest. water with a loosely tightened lid as the microwave must not be used dry)
• Culture dishes are coated depending on the cell type (1-2 h at 37°C or at 4°C over night)
• 1∙10⁵ cells per dish were seeded (also depending on the cell type) in 2 mL of culture medium
• Before starting imaging replace culture medium to colourless medium (wash with colourless medium first)

Preparation of the microscope

• Preheat the incubation chamber (2-3 h before starting the experiment)
• Connect CO₂-supply