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.