2. Matrigel for Invasion Study

Post date: Jul 9, 2012 4:41:31 AM

2.1 Aims

- Use as a thin gel layer (0.5mm), with cells plated on top.

2.2 Materials

- BD Matrigel™ Basement Membrane Matrix, 5 ml vial, 10mg/mL

Catalog Number: 356234

2.3 Methods

2.3.1 Preparation

- Once the Matrix is thawed, swirl vial

- Handle using sterile technique.

- Place thawed vial in sterile area, spray top of vial with 70% ETOH and air dry.

- Gently pipette using a pre-cooled pipette to ensure homogeneity.

- Dispense solution into appropriate aliquots, using pre-cooled tubes, and refreeze immediately.

o Aliquot: 0.5mL in eppendorf tubes

o Avoid multiple freeze thaws. DO NOT STORE IN FROST-FREE FREEZER.

2.3.2 Individual Use

- BD Matrigel Basement Membrane Matrix will gel rapidly at 22oC to 35oC. Thaw at 4oC on ice or fridge.

- Keep product on ice before use.

- Use pre-cooled pipettes, tips, and tubes when preparing BD Matrigel Basement Membrane Matrix for use.

- Gelled BD Matrigel Basement Membrane Matrix may be re-liquified if placed at 4°C on ice for 24-48 hours.

2.3.3 Dilution

- Thaw individual vial

- The concentration of the Matrigel is 10mg/mL

o For dilution, prepare coating buffer comprised of 0.01M Tris (pH 8.0), 0.7% NaCl.

o To do so, prepare a 10X Coating Buffer as follows:

§ Add 6.057g of Tris + 3.5g of NaCl to 500mL of distilled water

§ Filter using a 0.2 μm. Sterile filter unit.

§ Take 1mL Coating buffer and place in 9mL of ddH2O in a 15mL falcon tube

§ Cool the coating before to 4oC prior to use.

- Mix BD Matrigel matrix with Coating Buffer to bring concentration to 5mg/mL.

o i.e. 0.2mL matrigel + 0.2 mL coating buffer. This should be enough for at least 10 chambers.

- Thoroughly mix the coating solution containing BD Matrigel matrix by gently swirling, and then place the tube on ice.

2.3.4 Insert Coating

- Keeping culture plates on ice, dispense 30 μL in each well of a 96-well plate. Use sterile pre-cooled syringe.

o For 24-well plates, that would be a volume of 100uL

o Avoid air bubbles while pipeting the liquid into each insert.

If air bubbles get trapped in the wells, centrifuge at 300 x g for 10 minutes at 4°C

- Place plates at 37oC for 2 hours. Plates are now ready to use.