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Soft Agar Assay

Soft Agar Assay

Materials

  1. RK3E cells stably transfected with vector, EGFR and EGFR mutant, respectively
  2. Seaplque GTG low gelling temperature, BioWhittaker Molecular Applications: Cat# 50110
  3. Cell culture medium
  4. Trypsin-EDTA sulution
  5. D-PBS
  6. EGF solution
  7. Milli-Q H2O
  8. p-iodonitrotetrazolium violet
  9. Hemacytometer
  10. Microscope
  11. 4ml round-bottom, polycarbonate tubes
  12. 6-well tissue culture plates
  13. 100ml glass bottles
  14. Water bath
  15. Vortexer
  16. Tissue culture incubator
  17. Laminar flow hood
  18. Refrigerator

Procedure

  1. Use 6-well plates with 2 ml bottom and 2 ml top agarose/well.
  2. Prepare waterbath adjusted at 45oC in the hood. For 100 ml autoclave 0.9g Seaplaque agarose suspended in 20 ml distilled sterile water in 100ml glass bottle for 20 min, cool to 45oC. Add 80ml of growth medium for final agarose concentration of 0.9% and serum concerntration of 8%. Plate 2ml bottom agarose each well. Refrigerate leveled. In the meamtime, trypsinize and count cells, adjust to 1x105/ml growth medium
  3. Dilute leftover two-fold with growth medium yielding an agarose concentration of 0.45% in 37oC waterbath. Add trypsinized cells in 50ul aliquots to 4ml round-bottom, polycarbonate tubes (if this type of tubes are not available, the 15ml polypropanol centrifuge tubes can be used) at RT. Add EGF and 2ml top agarose, vortex, and plate on solidified bottom agar. Handle samples for one 6-well plate at a time. Refrigerate 30 min at 4oC and transfer to 37oC incubater.
  4. Refeed with 100ul of appropriate growth medium containing EGF once or twice a week depending on humidity.
  5. For staining dissolve 5mg p-iodonitrotetrazolium violet/ml PBS. Filter to remove insolubale material. Overlay dish with 0.25 ml/well. Incubate overnight at 37oC.
  6. Count colonies and record result.