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 45^oC 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 45^oC. 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 37^oC 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 4^oC and transfer to 37^oC 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 37^oC.
6. Count colonies and record result.