Gelatin degradation assay

Aim

Assess ECM degradation ability of cells.

Materials

1 mg/mL Fluorescein-gelatin

Preparation of fluorescein-gelatin solution

Dissolve 5 mg of fluorescein-gelatin in 5 mL of DDW

Voltex and heat at 50°C

Add sodium azide to get a final concentration of 2 mM (e.g., Add 100 uL of 100 mM sodium azide)

Aliquot into light-protected tubes and store at –20°C.

0.1 mg/mL Poly-L-lysine solution in DDW

PBS

0.5% Glutaraldehyde solution in PBS

70% Ethanol

Methods

Coat the glass surface with poly-L-lysine solution for 5 min at r.t.

Remove the poly-L-lysine solution and wash once with DDW, then dry up

Add 0.5% glutaraldehyde solution and incubate for 15 min at r.t.

Remove the glutaraldehyde solution and wash three times with PBS

Coat the glass with Fluorescein-gelatin solution for 10 min at r.t.

Put a 30 uL drop and cover it with a coverslip (φ12) (Fluorescein-gelatin is expensive. Please minimize its usage.)

Wash three times with PBS

Disinfect the gelatin surface with 70% ethanol for 30 min at r.t.

Wash with PBS

Add growth medium and incubate for 30 min at r.t.

Seed cells and incubate for appropriate period of time (Typically for 8~48 hours).

Cell density: e.g., 2x10^4 cells/mL

Fix and stain the cells if needed, and proceed to imaging.

Remarks

Method references