UCSF

Adhesion Assay

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% ethanol)
  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.