ECM Coating
Coating with Collagen, Fibronectin, Laminin and Matrigel* Matrices
Coating of membranes and plastic surfaces with extra cellular matrices (ECM) promotes cell attachment and monolayer formation. The following protocols describe coating with four types of ECM on Millicell-CM inserts. Although any Millicell membrane can be coated depending on specific protocols, only Millicell-CM membrane requires it for cell attachment.
There are also two procedures for coating the single-well plastic trays of Millicell-24 and Millicell-96 cell culture plates. These protocols are useful for growing cells on the plastic feeder trays in co-culture experiments.
*Matrigel is a registered trademark of Becton, Dickinson and Company
Coating with Collagen Type 1 Coating
Materials
- Millicell inserts and plates.
- 70% Ethanol (filter sterilized with a Millex-GP filter unit . Millipore cat. no. SLGP 033 RS; or a Stericup-GP filter cup . Millipore cat. no. SCG-P U11 RE)
- 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
- Dilute the collagen 1:4 in 70% ethanol (1 part collagen and 3 parts 70% ethanol) and vortex until the collagen is solubilized.
- Gather the applicable number of Millicell inserts or plates.
- Using a sterile pipette syringe, add the appropriate volume of the collagen/ethanol mixture to each well.
- Gently shake the cell culture plate until the collagen/ethanol mixture evenly coats the inside of the insert.
- 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.
- Seed the Millicell insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5×105 cells/cm2).
Volumes for Coating Millicell Devices
| 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
Additional Materials
- Human Fibronectin, 1 mg/mL (Catalogue Number: 08-102) reconstituted. Handle and store according to manufacturer's instructions.
Method
- Dilute the fibronectin 1:10 in the serum-free DMEM (1 part fibronectin in 9 parts DMEM) and vortex until fibronectin is solubilized.
- Gather the applicable number of Millicell inserts or plates.
- Using a sterile pipette syringe, add the appropriate volume of the fibronectin/DMEM mixture to each well.
- Gently shake the cell culture plate until the fibronectin/DMEM mixture evenly coats the Millicell-CM insert(s).
- 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
- Seed the Millicell insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5×105 cells/cm2).
Laminen Coating
Additional Materials
Laminin, 1 mg/mL (Catalogue Number: 08-125). Handle and store according to manufacturer's instructions.
Method
- Dilute the Laminin 1:10 in DMEM (1 part laminin in 9 parts DMEM) and vortex until the laminin is solubilized.
- Gather the applicable number of Millicell inserts or plates.
- Using a sterile pipette syringe, add the appropriate volume of the laminin/DMEM mixture to each well.
- Gently shake the cell culture plate until the laminin/DMEM mixture evenly coats the Millicell inserts.
- 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.
- Seed the Millicell insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5× 105 cells/cm2).
Matrigel Coating
Additional Materials
Matrigel CoatingMethod
- Dilute Matrigel according to manufacturer’s instructions.
- Gather the applicable number of Millicell inserts or plates.
- Using a sterile pipette syringe, add the appropriate volume of the Matrigel mixture to each well.
- Gently shake the cell culture plate until the Matrigel solution evenly coats the Millicell insert.
- 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.
- Seed Millicell-CM insert with appropriate cell density (for example, MDCK cells: approximately 5×104 to 5×105 cells/cm2).
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