Coating 6-well or 12-well Plates with Collagen

Youssef Farhat, Written 9/26/12, Last updated 5/6/14

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Introduction

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 [1].  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.

Materials

  • 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)

  1. Under the culture hood, prepare 200 mL of 1.25X DPBS:
    1. Combine 5.6 mL of 10X PBS with 194.4 mL of 1X DPBS.
  2. 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:
    1. 266 µL of NaOH (0.1 M)
    2. 158 µL of 10X PBS
    3. 12.6 mL of 1X DPBS
    4. 1.16 mL of Rat Tail Collagen solution
  3. Mix these reagents thoroughly by pipetting up and down or closing the lid of the container and inverting the centrifuge tube several times.
  4. Take 1 mL of collagen into a 1.5 mL Eppendorf tube and test the pH with a pH strip.
    1. 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.
    2. 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.
  5. 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.
    1. 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.
  6. 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…

  1. Carefully transfer the plate from the incubator to the culture hood.
    1. Note: Be careful not to shake or disturb the delicate collagen gel while transferring.
  2. 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…

  1. 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).
  2. Leave plates under the hood for 30 min so that the salt precipitates fully dissolve.
  3. Remove the water by slow, careful pipetting to avoid scratching or damaging the collagen coating.
  4. Repeat the previous three steps (1-3) in order to wash two more times.
  5. Remove the distilled water from the last wash.
  6. 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).

References

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

Farhat, Y. “Coating 6-well or 12-well Plates with Collagen.” The Protocol Place. September 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com/>

2 Responses to Coating 6-well or 12-well Plates with Collagen

  1. This protocol was updated on 3/20/13 and again on 4/6/13. In the previous versions, a different type of collagen was used, and a different procedure for coating. The present version has significant changes that have made this protocol work better.

    1) switching from bovine collagen (“PureCol”) to rat tail collagen
    2) gelling the collagen in the incubator before setting in the hood to dry to a film overnight
    3) the order in which the reagents are combined was altered so that the collagen gets added last. In the new method, potential effects from drastically changing the collagen’s pH is avoided by adding the collagen to a buffered solution (pH is buffered by the PBS).

  2. Youssef Farhat

    Important changes made to this protocol on 4/22/13

    - Clarified the target pH of the neutralization for steps 4A and 4B to be 7.0-7.5 for the first day of the protocol.
    - Added a 30 min incubation step to the washing of the collagen coated plates (now step two of the last day of the protocol).

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