Clonogenic Survival Assay
Day 1 – Fast forward transfection:
Plate 5×105 U2OS in 6-well plates (in 2 mls media)
Optimem: 82 μL
HiPerfect: 12 μL
siRNA: 6 μL of 2μM stock
Incubate 5-10 min at RT and add dropwise to cells
Day 2 – Split into 100 mm dishes
Day 3 – Count cells and plate in 60 mm dishes x 3 for each dose
Unt – 200 cells/dish
3gy – 2000 cells/dish
5gy – 10,000 cells/dish
Day 4 – Treat IR – 3gy and 5gy
Incubate for 10-14 days until colonies grow large enough to easily count
Stain with methylene blue:
50% Methanol
48% H2O
2% methylene blue
Aspirate media from dishes
Cover bottom of plate with staining solution (~ 1mL) for at least 1 min.
Decant staining solution
Wash plates in 4 L beaker in sink with water running
Allow plates to air dry before counting colonies
* Protocol can be modified to treat with IR 48h after siRNA transfection by plating morning of day 3 then treating in the evening or by plating directly from 6 well plate on day 2 then treating day 3.