15. Putting down fibronectin and collagen - dfierros/cohen_lab GitHub Wiki

Fibronectin (“no coverslip” procedure)

• For one plate, use 12.5 microliters fibronectin, 237.5 microliters DI water (250 ml total)
• Pipet water into little blue capsule
• Pipet fibronectin into capsule (if more than 25 microliters can put this in first)
• Use pipet to mix (draw up and down)
• Pipet into center of circular portion of dish and tilt to cover whole circle
• If thin layer, may use parafilm to prevent evaporation
• Place in incubator for 30 min
• Tilt dish and aspirate remaining solution from edge of circle
• Do NOT touch aspirator to bottom of dish
• Wash 3 times with DI water (just pour into dish, swirl, dump out)
• Aspirate off remaining water
• Leave upside down on cover to dry (push to back of hood so its out of the way)

Collagen

• Same as fibronectin but with PBS in the first step
• Ratio is 5 microliters collagen, 95 microliters PBS
• So add PBS to a tube, then add collagen to that tube (large, then small)
• Mix by pipetting up and down
• Lay down a cover slip on top with tweezers (bunsen burner the tweezers first)
• Incubate for 30 minutes
• Wash/Aspirate (50 ug/mL concentration)