Adhesion Assay Protocol

Materials to be prepared beforehand: 1) Washing Buffer--0.1% BSA in medium (DMEM or RPMI) 2) Blocking Buffer--0.5% BSA in medium (DMEM or RPMI) 3) Laminin-1 10-12 μg/ml or FN 20 μg/ml 4) 96-well-plate 5) Crystal violet (5mg/ml in 2% Ethnol) 6) 1% SDS in H2O 7) 4% paraformaldehyde Procedures: 1) Coat 96-well-plate with Laminin-1 or FN at 37 oC for 1 hr or at 4 oC O/N. Leave some wells uncoated as negative control. 2) Wash with washing buffer for 2 times. 3) Block plates with blocking buffer at 37 oC in CO2 incubator for 45-60 minutes. 4) Wash with washing buffer. 5) Chill the plates on ice. 6) Count cell to 4 X 105/ml. Add 50 μl cells in each well. 7) Incubate in CO2 incubator at 37 oC for 30 minutes. 8) Shake the plate at 2000 rpm for 10-15 seconds. Wash with washing buffer 2-3 times. 9) Fix with 4% paraformaldehyde. Incubate at RT for 10-15 minutes. 10) Wash with washing buffer. 11) Stain with Crystal Violet for 10 minutes. 12)Wash with water. 13) Turn the plates upside down. Let the plates dry up completely. 14) Add 2% SDS. Incubate at RT for 30 min. 15) Read plate at 550μm.