EC Migration Assay

 2H11 Migration Assay Protocol

Mayo Lab (Dec. 2011)

Sterling Mikkelson

Materials:

·         Ibidi 24-well culture inserts (http://www.ibidi.de/products/disposables/E_80XXX_CI_StemCell_family.html)

·         Coating Matrix (Gelatin 0.2%, Collagen 0.01%, Fibronectin 1.0%)

·         RPMI 1640 (10% FBS, 1% Pen-Strep)

·         0.25% Trypsin

·         Hemacytometer

Procedure:

Ø  Day 1

·         Coat each well with desired matrix and incubate for 1 hr. prior to preparing cells for plating

·         Aspirate media from stock 2H11 flask

·         Add 5 ml 0.25% Trypsin and incubate for 5 min.

·         Add 6 ml RPMI media to flask containing trypsin, pipette up and down 3-5 times, and add to 15 ml centrifuge tube.

·         Spin at 1500 rpm for 5 min.

·         Aspirate supernatant and add 3-6 ml media (depending on pellet size)

·         Count cells using hemacytometer (ideal amount of cells should be between 15-50 cells per square)

·         Determine cell concentration (Will need 30,000 cells/ 70 ml)

·         Remove coated culture wells from incubator and aspirate coating.

·         Plate calculated cell concentration (30,000 cell/ 70 ml) into each well and incubate 18-24 hours

Ø  Day 2

·         Prepare compound of interest at desired concentration in a total volume of 400 ml

·         Remove 24-well plate from incubator and carefully remove media in each well using a 200 ml pipettor

·         Using a tweezer, grasp the top corner of the insert and pull up and towards you to remove

·         Slowly, add solution (400 ml total volume) to each well, making sure wells are evenly coated

·         Use inverted microscope at Biomedical Image Processing Lab (BIPL)

·         Under 4X objective magnification, capture migration activity at several time point (0 - 24hr)