Protocol for Collagen Invasionassay

Collagen gels for invasionassay:

Prepare everything on ice (also cooled tips if possible):

1.2 ml collagen G (Biochrom 4 mg/ml)
1.2 ml collagen R (Serva 2 mg/ml)
270 µl 10x DMEM (Biochrom 4.5 g/l D-Glucose, without NaHCO3/L-Glutamine/Na-Pyruvat)
270 µl NaHCO3 (23 mg/ml in H2O, M=84.01g/mol)
43 µl NaOH (Sigma 1.0 N solution)

mix well but carefully

→ 1.2 ml per 3.5 cm petri dish

→ polymerisation in incubator for 2 h

→ add 2 ml PBS

→ seed 50,000 cells on top the day after

→ wait for 3 days

→ fix the cells using 2 ml glutaraldehyde (2.5 % in PBS) for 20 min

→ wash with PBS

→ stain with 5 µl Hoechst for 20 min

→ wash with PBS