STEWART LABORATORY

 

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Soft Agar Assay

 

 

MAKE 2.4% NOBLE AGAR

(Difco Cat #214230)

 

      Mix:

 

         100mL ddH2O

 

         2.4 g agar

 

      Microwave when you are ready to begin (we no longer autoclave the agar)

 

 

MAKE 0.6% MEDIA-AGAR MIX FOR THE BOTTOM LAYER

(All ingredients should be at 37C. The agar should be freshly microwaved and at 60C)      

 

      Mix:

 

         100 mL 2X DME

 

         2 mL Pen/Strep

 

         20 mL IFS

 

         50 mL molten 2.4% Noble agar

 

         28 mL ddH2O

 

      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.

 

 

PREPARE THE TOP LAYER MIXTURE

(Do not add 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 37C to prewarm

 

         Mix:

 

        2X DME 12.5 mL

 

        IFS 2.5 mL

 

        Pen/Strep 0.25 mL

 

        ddH2O 3.5 mL

 

 

LABEL, LIFT AND COUNT CELLS

      For each cell line there should be 3x 15 mL conical tubes labeled 105, 104, and 103. Each of these should have the cell name on the tube

 

      Lift two cell lines and pass through a 100μm cell strainer

 

      Count cells

 

      Re-suspend the cells at 1 x 105/mL (make 5 mL)

 

 

SET UP DILUTIONS

 

      Add 1.0 mL of D10 (DME + 10% IFS) 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 (youre not done with this tube yet - see below)

 

      Add 1.9 mL D10 and 100 μL from the 104 tube to the 103 tube

 

      Remove 2.9 mL from the 104 tube (this should leave you with 7 mL total)

 

 

COMBINE AND PLATE MEDIA

 

      Melt the 2.4% Nobel agar that has been prepared in ddH2O (you made this earlier when you made the bottom layer)

 

      Add 6.2 mL Nobel agar to one of the 50 mL conical tubes that contains the DME mix, and mix by inverting several times. Check to be sure that the
contents of the tube are cool enough (you should be able to touch it to the inside of your wrist and it should be comfortably warm)

 

 

Now you need to work fast but avoid air bubbles!!!!!!

 

      Add the following amounts of agar/media mix to each tube:

 

         Add 2 mL to the 103 and 105 tubes

 

         Add 7 mL to the 104 tube

 

      Mix by inverting several times

 

      Plate 4 mL of mix on each corresponding plate

 

      Allow the plates to sit 30-60 min until they are solidified and then carefully transfer them to the incubator

 

      Go back to adding agar/media mix for the next cell line

 

 

For long-term experiments you can feed the plates if they are looking dry or yellow with a 0.3% agar mix