Eccles:Collagen Coating of Cell Culture Vessels

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Collagen Coating of Cell Culture Vessels

  • Pipette collagen solution (0.05ug/ul collagen in 1x PBS) onto surface and swirl to disperse as you would with cells.
  • Top up the volume with more 1x PBS if required (96, 6 wells and T75 flasks) so that the surface remains covered with liquid for the whole 2h.
  • Leave collagen solution on plate surface for 2h (can be left in cell culture hood or incubator).
  • Aspirate off liquid and rinse 2x in 1x PBS (in an ideal world. FYI when I first began working with cells that were grown on collagen coated surfaces I had so many cells to deal with that it wasn't practical time-wise to do this. I aspirated off liquid and eliminated the rinse. My rationale was that the stock collagen (in 0.1M acetic acid) has been diluted 20 fold before use on plates/flasks and then the excess liquid is aspirated off therefore any remaining acetic acid would be very minimal). All of my collagen coating has been carried out in this manner so it is consistent. Add media to keep collagen moist. Add cells as usual or vessel can be kept in cell culture incubator until ready to add cells.

Vessel Size Flask/Well Surface Area (cm2) Media Volume Volume Collagen Solution (0.05ug/ul) to Achieve ~6ug/cm2
96 well plate 0.28 0.2ml 33ul
24 well plate 2.0 0.5ml 300ul
6 well plate or 35x10mm round dishes 9.6 2ml 1.1ml
T25 flask 25 5ml 2.9ml
T75 flask 75 20ml 8.8ml
T175 flask 175 50ml 20.6ml

Collagen Solution (0.05ug/ul)

  • Stock collagen is at 1ug/ul in 0.1M acetic acid (Sigma #C8919)
  • Dilute collagen in 1x PBS to 0.05ug/ul ready for use (store at 4 degrees C)

Collagen Stock (1ug/ul) 1x PBS
Volume (to make 50ml) 2.55ml 47.45ml