BioProtocols--cell8

Migration Assay on Endothelial Cells (ECs)

1. ECs, fed 24 hrs before experiment, 70-80% confluent at time of experiment.

2. Transwells (8um, 6.5 mm, polycarbonate, Costar, cat #3422), coated on lower side with 10 ug/ml fibronectin or vitronectin or other matrix in PBS (50 ul per well), overnight at 4oC or until dried (~2 hrs) in tissue culture hood. Control is coated with 0.5% fatty acids free BSA in PBS.

Use at least triplicate for each condition.

Random migration control measured in wells with both top and bottom coated with same matrix protein.

Place into outer well 500 ul RPMI 1640/0.5% BSA (supplemented cations where indicated), with or without FBS.

3. Collect cells, count and calculate total number. Centrifuge at 900 rpm for 5 minutes at 4oC, rinse once and then resuspend in 0.5%BSA/RPMI to give concentration of 2x105 cells/ml. Then aliquot cells to 50 ml plastic tubes and concentrate and provide the different conditions.

4. Dispense 100 ul = 20,000 cells per sample into each appropriate upper chamber. Allow to migrate at 37oC for 2-24 hrs. Once completion of incubation, view briefly under microscope before fixation.

5. To fix: dab corner of transwell on paper towel, wipe cells off the top of membrane well with flattened-tip Q-tip, paying extra attention to the edges of the membrane with the walls

6. If staining w DiffQuick, proceed w stain per manufacturer’s instructions.

7. If doing crystal violet stain, drop into a new outer well with 250 ul 0.1% crystal violet in 100mM Borate pH 9.0, 2% Ethanol (make fresh daily; careful, flies everywhere and stains surfaces and clothes). Aspirate medium from original wells. Incubate total 5-20 minutes at room temp then rinse (dab corner on paper towel, rinse well in dH2O, and wipe outer surface with kimwipe, careful not to touch lower part of the membrane, wipe well the upper side of the membrane and inside walls of the transwell with Q-tip).

8. View under bright field inverted microscope to assess migration. Count the cells in all wells. Photograph at low power and high power for documentation and for quantification in photoshop.

9. If used crystal violet, can elute dye in 250 ul 10% acetic acid for 5 min, and then measure OD of 200 ul in 96 well plate @ OD600.