Collagen coating protocol

Neuvitro Corporation owns the leading technology to provide professional coating with the broadest coverage of substrates, such as PDL, PLL, PLO, Collagen, Gelatin, Laminin, Fibronectin, PDL+Laminin, PDL + Fibronectin, PLO+Laminin, PLO+Fibronectin, PDL+Laminin+Fibronectin, etc. The majority of coated German coverslips available from Electron Microscopy Science, ThermoFisher Scientific, and VWR is actually manufactured by Neuvitro Corporation.

Tips for handling collagen solution in coating:

Unlike most of othe coating substrates, collagen i and iv remain as soluble at 4C degree and begin their solidification process at room temperature. At 37C in cell culture incubator, collagen molecules solidify quickly.
The PH value of collagen solution plays an important role for collagen solidification. To keep collagen soluble, you must reduce the PH value low to 5 or less. To solidify collagen solution, you can add some 0.1mM NaOH to collagen solution. The more your add the faster it become collagen gel.
Using collagen i to embed explant tissue on plates, slides, or coverslips. A 3-D structure is formed by solidified collagen i to wrap the tissue explant inside. Oxygen and nutrition are supplied to the embedded tissue explant through solidified collagen. You can control the size of communication channel by altering the concentration of collagen i.
Using collagen i or iv to coat a thin layer on plates, slides, or coverslips for dispersed cell culture. Cells are exposed to culture medium directly. Only half of cell culture are anchored by collagen coated surface.

Collections of different collagen coating protocols:

Santa Cruz Biotechnology recommended collagen coating protocol

Dilute collagen to 50ug/ml with 0.01M HCl.

Add sufficient diluted collagen to coat dishes, 5-10ug/cm surface.

Incubate the coating for 1 hour at room temperature.

Remove remaining solution and rinse wells with PBS or culture medium to remove acid.

Use the plate immediately, or air dry the plates and store the coated plate at 2-8°C

CellSeed recommended collagen coating protocol

Prepare collagen solution 500ug/ml with 0.02M acetic acid.

Add 1ml of the diluted collagen solution to 3.5cm diameter dish to cover all area.

Incubate dish 1 hour at 37°C incubator.

Wash dish with pre-warmed PBS.

Invitrogen recommended collagen coating protocol

Determine the volume of collagen solution needed.

Dilute collagen to 50ug/ml in 0.02M acetic acid to the needed volume.

Add diluted collagen, 5ug/cm² surface area, to dish or plate.

Incubate for 1 hour at room temperature.

Remove remaining collagen solution.

Rinse dish 3 times with PBS or culture medium.

Use dish immediately or air dry for future use.

Collagen coating protocol posted at University of Tennessee

Dissolve collagen 2mg/ml in 0.2% acetic acid.

Filter the collagen through 5um filter.

Filter it again through 0.45um filter, then store at 4°C.

When coating, dilute the stocked collagen 1:5 in tissue culture grade water.

Dispense diluted collagen solution to dishes and sit overnight.

2ml / 35mm Petri dish

3ml / 60mm Petri dish

5ml / 100mm Petri dish

4ml / T25 flask

12ml / T75 flask

0.5ml / 24 transwell

Remove remaining collagen solution and air-dry.

Millipore recommended collagen coating protocol

Dilute collagen 1:4 in 70% ethanol and vertex it to mix.

Add the diluted collagen mix to plates in appropriate volume:

50ul/24 well plate, 150ul/12 well plate, 400ul/6 well plate.

Shake the plate to cover all area evenly.

Air dry the plates 3 hours to overnight.

AMSbio recommended collagen coating protocol

Prepare collagen solution 0.8mg/ml cell culture grade water on ice to prevent collagen from gelling.

Remove pre-wash PBS and add the collagen.

Leave the plate overnight in hood to dry.

Wash in PBS or culture medium.

Seed cells.

NUNC recommended collagen coating protocol

Prepare collagen solution 10ug/ml in 10% IPA, 0.1M acetic acid.

Apply collagen coating solution.

Incubate overnight at 4-8°C.

Remove remaining solution and air dry overnight at room temperature.

Sigma-Aldrich recommended collagen coating protocol

Prepare collagen stock solution 0.1%(w/v) in 0.1M acetic acid.

Prepare working solution by dilute 10-fold to 0.01%

Apply 6-10ug/cm² surface area and incubate several hours at room temperature or overnight at 2-8°C.

Remove remaining solution and air dry overnight at room temperature.

Commercially available collagen coated coverslips

Collagen, a component of extracellular matrix also binds to fibronectin and laminin, enhance cell attachment in culture.

There are mainly two types of cultures using collagen to enhance attachment:

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