ALI transwell filters

Culturing cell lines on transwell-style permeable supports:
  • For primary cells, we use Corning 3470 (PET, clear, 0.4 µm pore, 0.33 cm2 surface area; 24 well plate)
  • For cultured cells (16HBE, BEAS2B, H292, CFBE, A549, RPMI2650), we use Greiner Thincerts 662461 (PET, transparent, 0.4 µm pore, 24 well plate)
  • For Calu-3 cells, we use Greiner Thincerts 665641 (PET, transparent, 0.4 µm pore, 12 well plate)

Transwells must be coated prior to use.  Coat transwells (100 µL for 0.33 cm2 transwell and 200 µL for 1 cm2 transwell) for >2-3 hrs (preferably overnight) at 37 °C.  Aspirate excess fluid and wash once with PBS.  

Coating solution components
  • Bovine Type I Collagen (Corning 354231) 3 mg/ml liquid (open only in hood, seal tightly after use; store at 4 °C)
  • Human Fibronectin (Corning 354008) 1 mg powder (single use; open in hood; Cell Center item # 1224)
  •  BSA (Sigma A4919); Make 1mg/ml solution of BSA in MEME and filter sterilize in the hood; freeze aliquots at -20°C.   
  • Making coating solution: 
    • 100 ml MEME
    •   Dissolve 1 mg fibronectin with a few mls of the MEME
    • Add 1 ml BSA solution
    •  Filter sterilize
    • Add 1 ml collagen after sterilization
    • Final concentrations: 30 µg/ml collagen, 10 µg/ml fibronectin,  10 µg/ml BSA
    • Be careful, as this solution has no antibiotics
    • No need to warm this solution before coating transwells
    • Store at 4 °C in the dark.