Youssef Farhat, Written 9/26/12, Last updated 5/6/14
Collagen is the most abundant protein of the body, and cells are surrounded by it in almost every tissue and organ. Therefore, coating collagen onto culture dishes provides a more realistic in vitro environment for the study of several cell types.
This protocol details one method for coating collagen onto 6-well or 12-well plates at a density of 50 µg/cm2 that was adapted from a Cancer Research paper . Each step must be carried out using sterile technique and sterile reagents in order to avoid contaminating the culture environment.
The following protocol is for coating only one 6-well or 12- well plate, so the volumes indicated must be scaled up or down as necessary.
- Collagen I, rat tail, 100 mg (BD Biosciences, #354236)
- Sterile DPBS (Gibco, #14190)
- Sterile 10X PBS (Gibco, #70013)
- Sterile distilled water (Gibco, #10977)
- Sterile NaOH (Mix 399 mg NaOH into 100 mL of deionized H2O and sterile-filter)
- 6-well culture dishes (BD Falcon, #353046) or 12-well dishes (#353043)
- pH strips (EMD, Cat. # 9588)
- General lab equipment, such as a culture hood, 50-mL centrifuge tubes micropipettors, micropipette tips, etc.
Procedure (for one 6-well plate or 12-well plate)
- Under the culture hood, prepare 200 mL of 1.25X DPBS:
- Combine 5.6 mL of 10X PBS with 194.4 mL of 1X DPBS.
- Under the culture hood, prepare a collagen coating solution (300 μg/mL) by combining the following into a 50-mL centrifuge tube or similar sterile container in order:
- 266 µL of NaOH (0.1 M)
- 158 µL of 10X PBS
- 12.6 mL of 1X DPBS
- 1.16 mL of Rat Tail Collagen solution
- Mix these reagents thoroughly by pipetting up and down or closing the lid of the container and inverting the centrifuge tube several times.
- Take 1 mL of collagen into a 1.5 mL Eppendorf tube and test the pH with a pH strip.
- If the pH is too high/basic (e.g. >7.6), add 0.2 mL of collagen I, 0.8 mL of 1.25X DPBS, and repeat steps 3 & 4 until a pH of 7.0-7.5 is obtained.
- If the pH is too low/acidic (e.g. <7.2), add 0.15 mL of collagen I, 0.05 mL of NaOH, add 0.8 mL of 1.25X DPBS, and repeat steps 3 & 4 until a pH of 7.0-7.5 is obtained.
- Add 1.58 mL of collagen coating solution to each well of the 6-well plate (using a micropipettor, pipette 792 µL twice), or 0.634 mL of collagen coating solution to each well of the 12-well plate.
- Note: It’s easiest to add the collagen to the center of each well (not the side). After finishing all the wells, gently swirl the plates in a circular motion to spread the collagen evenly across the surface of the wells.
- Incubate the 6-well or 12-well plate in a culture incubator set to 37°C overnight in order to allow the collagen to fully polymerize.
The next day…
- Carefully transfer the plate from the incubator to the culture hood.
- Note: Be careful not to shake or disturb the delicate collagen gel while transferring.
- Leave the plate uncovered in the culture hood several hours or overnight so that the collagen dries down into a thin film.
The next day…
- Wash the collagen coated wells by slowly adding sterile distilled water to each well (3 mL/well for 6-well plate, 1 mL/well for 12-well plate).
- Leave plates under the hood for 30 min so that the salt precipitates fully dissolve.
- Remove the water by slow, careful pipetting to avoid scratching or damaging the collagen coating.
- Repeat the previous three steps (1-3) in order to wash two more times.
- Remove the distilled water from the last wash.
- Seed the plate immediately with cells, or leave the plate in the culture hood uncovered to dry for later use (it should take <1 h to dry).
1. Aznavoorian S, Moore BA, Alexander-Lister LD, Hallit SL, Windsor LJ, et al. (2001) Membrane type I-matrix metalloproteinase-mediated degradation of type I collagen by oral squamous cell carcinoma cells. Cancer Res 61: 6264-6275.
Citing this Protocol