Soft Agar Assay

1. Make 0.6% media-agar mix for the bottom layer.

        To make 0.6% agar mix the following components (this makes 200 ml):

1. 2X DME 100 ml
2. IFS 20 ml
3. Pen/Strep 2 ml ddH2O
4. 28 ml
• warm components to 37°C
• Add 12.4 g of Noble agar to 100 ml ddH₂O and microwave to melt (do not over microwave or you will lose too much volume). Microwave just long enough to completely melt the agar.
• Add 50 ml of agar to the prewarmed mixture that you just prepared.
• swirl to mix avoiding air bubbles.
• add 4 ml of this mix to each 6 cm2 plate
• allow to completely cool while you prepare your cells.

2. Prepare the top layer mixture with all of the listed components EXCEPT the Noble agar.

You will need one 50 ml conical for every two cell lines that you are testing. Prepare the following mix and place tube(s) at 37°C to prewarm.

Mix:

3. 2X DME 12.5 ml
4. IFS 2.5 ml
5. Pen/Strep 0.25 ml
6. ddH₂O 3.5 ml

7. Label tubes. For each cell line there should be 3-15 ml conical tubes labeled with 105, 104, or 103 and each of these should have the cell name on the tube.
8. Lift two cell lines and pass through a 100μm cell strainer.
9. Count cells.
10. Resuspend the cells at 1 x 105/ml (make 5 ml).
11. Set up dilutions as follows:
    • Add 1.0 ml of D10 and 1.0 ml of the 1 x 105/ml dilution to the 105 tube.
    • Add 9.5 ml D10 and 500 μl from the 1 x 105/ml to the 104 tube and mix by inverting (you’re not done with this tube yet - see below).
    • Add 1.9 ml D10 and 100 μl from the 104 tube to the 103 tube.
    • Now remove 2.9 ml from the 104 tube (this should leave you with 7 ml total)
12. Now melt the 2.4% Nobel agar that has been prepared in ddH2O (you made this earlier when you made the bottom layer).
13. Add 6.2 ml Nobel agar to one of the 50 ml conical tubes and mix by inverting several times.
14. Check to be sure that the contents of the tube are cool enough (you should be able to touch the inside of your wrist and it should be comfortably warm.
15. Now you need to work fast but avoid air bubbles!!!!!!
16. Add the following amounts of agar/media mix to each tube:
    1. Add 2 ml to the 103 and 105 tubes
    2. Add 7 ml to the 103 tube.
    3. Mix by inverting several times.
17. Plate 4 ml of mix on each corresponding plate.
18. Allow the plates to sit 30 to 60 minutes until they are solidified and then carefully transfer them to the incubator.
19. Go back to step 4 for the next two cell lines.
20. For long-term experiments you can feed the plates if they are looking dry or yellow with a 0.3% agar mix.