I am using Matrigel Corning for cell invasion assay. How can I make a thin Matrigel layer?
Dear Soumyadip,
Always use whatever basal media the cells grow in (without FBS). We use 15-30% matrigel for invasion assays. Are you doing it in a scratch-wound format or in the traditional hanging well inserts with a membrane (use 8um holes for cancer cells!).
Steve
Always use the Tris-NaCl coating buffer!Make your buffer, and filter-sterilize it. Keep it on ice. Thaw the ECM on ice. Add the appropriate amount of ECM gel (final concentration 200-300 micrograms/mL) to about 2.0mL of Tris-NaCl. Add 100 microliters of the buffer/ECM mixture to the wells, taking care not to get it on the sides of the wells and make sure there are no air bubbles. Incubate at 37 degrees celsius for 2 hours, and then pipet off the excess liquid, taking care not to disturb the gel layer you have just created. Then you can add your medium/chemoattractant to the wells, and add your cells to the inserts. Incubate overnight, and remove medium and cells from the apical side of the membrane. Fix cells in methanol on ice for 30 minutes, and stain as you wish.
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