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

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

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

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).