ECM Coating Protocols for Millicell®

Coating of membranes and plastic surfaces with extra cellular matrices (ECMs) promotes cell attachment and monolayer formation. We have developed protocols for four types of ECMs on Millicell®-CM inserts. They are also useful for growing cells on plastic feeder trays in co-culture experiments.

Collagen Type 1 Coating

Materials

  • Millicell® inserts and plates.
  • 70% Ethanol
  • Rat Tail Collagen, Type 1, approximately 3 mg/mL (Millipore cat. no. 08-115)
  • Sterile pipette syringes and tips

Note: An alternate collagen source (Type I) of suitable concentration diluted in 0.01 N HCl or acetic acid may also be used.

Method

  1. Dilute the collagen 1:4 in 70% ethanol (1 part collagen and 3 parts 70% ethanol) and vortex until the collagen is solubilized.
  2. Gather the applicable number of Millicell® inserts or plates.
  3. Using a sterile pipette syringe, add the appropriate volume of the collagen/ethanol mixture to each well.
  4. Gently shake the cell culture plate until the collagen/ethanol mixture evenly coats the inside of the insert.
  5. Air dry inserts in a laminar flow hood. Leave cell culture plate cover ajar to allow airflow and prevent condensation. Note: Although drying typically takes anywhere from 3 hours to overnight, overnight drying is recommended.
  6. Seed the Millicell® insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5×105 cells/cm2).

Volumes for Coating Millicell® Device

Coating (24-well) (12-well) (6-well) (MIllicell 24) (Millicell 96)
Collagen (Collagen/Ethanol mix) 50 µL 150 µL 400 µL 100 µL 25 µL
Fibronectin* (Fibronectin/DMEM mix) 100 µL 300 µL 700 µL 200 µL 50 µL
Laminin* (Laminin/DMEM mix) 100 µL 300 µL 700 µL 200 µL 50 µL
Matrigel* (Matrigel/H2O solution) 100 µL 300 µL 700 µL 200 µL 50 µL

*Can be used on any Millicell® membrane
*Matrigel is a registered trademark of Becton, Dickinson and Company

Fibronectin Coating

Materials

  • Millicell® inserts and plates.
  • Sterile pipette syringes and tips

Human Fibronectin, 1 mg/mL (Catalogue Number: 08-102) reconstituted. Handle and store according to manufacturer's instructions.

Method

  1. Dilute the fibronectin 1:10 in the serum-free DMEM (1 part fibronectin in 9 parts DMEM) and vortex until fibronectin is solubilized.
  2. Gather the applicable number of Millicell® inserts or plates.
  3. Using a sterile pipette syringe, add the appropriate volume of the fibronectin/DMEM mixture to each well.
  4. Gently shake the cell culture plate until the fibronectin/DMEM mixture evenly coats the Millicell®-CM insert(s).
  5. Air dry inserts overnight in a laminar flow hood. Leave cell culture plate cover ajar to allow airflow and prevent condensation. Note: Although drying typically takes anywhere from 3 hours to overnight drying is recommended
  6. Seed the Millicell® insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5×105 cells/cm2).

Laminin Coating

Materials

  • Millicell® inserts and plates.
  • Sterile pipette syringes and tips

Laminin, 1 mg/mL. Handle and store according to manufacturer's instructions.

Method

  1. Dilute the Laminin 1:10 in DMEM (1 part laminin in 9 parts DMEM) and vortex until the laminin is solubilized.
  2. Gather the applicable number of Millicell® inserts or plates.
  3. Using a sterile pipette syringe, add the appropriate volume of the laminin/DMEM mixture to each well.
  4. Gently shake the cell culture plate until the laminin/DMEM mixture evenly coats the Millicell® inserts.
  5. Air dry inserts overnight in a laminar flow hood. Leave the cell culture plate cover ajar to allow airflow and prevent condensation. Note: Although drying typically takes anywhere from 3 hours to overnight, overnight drying is recommended.
  6. Seed the Millicell® insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5× 105 cells/cm2).

Matrigel Coating

Materials

  • Millicell® inserts and plates.
  • Sterile pipette syringes and tips

Method

  1. Dilute Matrigel according to manufacturer’s instructions.
  2. Gather the applicable number of Millicell® inserts or plates.
  3. Using a sterile pipette syringe, add the appropriate volume of the Matrigel mixture to each well.
  4. Gently shake the cell culture plate until the Matrigel solution evenly coats the Millicell® insert.
  5. Air dry inserts overnight in a laminar flow hood. Leave cell culture plate cover ajar to allow airflow and prevent condensation. Note: Although drying typically takes anywhere from 3 hours to overnight, overnight drying is recommended.
  6. Seed Millicell®-CM insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5×105 cells/cm2).

Materials