I. INTRODUCTION

The soft agar experiment described below, you will compare the same four cell cultures with respect to their anchorage dependence. It is well known that, in general, normal cells are obligatorily anchorage dependent whereas malignantly transformed cells, being anchorage independent, can be grown unattached to a growing surface or support, or even suspended in liquid media. Growth suspended in agar is a convenient means to test whether cells are anchorage dependent or not.

Growth and Characterization Using Soft Agar

II. MATERIALS

Media:
a. 2X DME-F12* + 10% FBS
*This is made by adding only half the water required to make this medium.

b. DME-F12 + 5% FBS

Cell Lines: Yours

Waterbath at 44^o C

Difco Bacto-Agar (prepared as described below)

Part I: Preparation of agar stock and agar base for dishes:

1. Dissolve Difco Bacto-Agar in double distilled water (12.5 grams per 1000 ml of water [1.25%]). To dissolve agar, boil in a double boiler or place in a microwave oven. Dispense into convenient aliquots and sterilize by autoclaving. Store at room temperature. (This step will be carried out for you.)

2. Melt agar (How do you melt agar?) and maintain at 44^o in waterbath.

3. Warm 2X medium containing 10% fetal bovine serum to 44^o .

4. In this experiment you will have to work quickly but as carefully as always. Quickly, because agar melts at 100^oC, but it doesn’t solidify until it reaches approximately 37^oC. However, the agar is maintained at 44^oC because, although this temperature will kill cells, if they are maintained at this temperature for long, the solution cools quickly below the toxic level at room temperature. So, unless you work quickly, the agar will solidify in the pipet as you are using it. The hint below will help you greatly so use it!.Mix melted agar and 2X medium 1:1 [yielding 0.625% agar and 1X medium] to a final volume of twice volume for the number of plates to be used (in this case, 4 plates at 4.0ml per plate or 32ml) and pipette 4 ml per dish into the plates (P-60's). (Hint: 1) For convenience, use 34 ml. I.e. mix 17ml 2x media + 17ml melted agar = 34ml of 0.625% agar.  16ml will be used to plant the bottom layer.  2)Keep the flask of agar in a volume of 44^o water to help maintain the agar in the fluid state.) Tip plate to spread agar evenly and let harden for 10-30 min. Place in incubator after agar hardens. Place remaining 1:1 mixture (16) in 44^o bath to maintain in the liquid state.

  Part 2: Preparation of Cells and Plating in 0.3% Agar: [This part may seem confusing but it will be discussed fully prior to you actually doing the experiment.]

1. Trypsinize cells:  Adjust cell number, in complete 1X medium, to 1 x 10⁵ cells/ml and treat as follows:

Stock = 1 x 10 ⁵ cells/ml. Dilute 1.0ml cells + 4.0ml medium (A dilution of ___to___)= 2.0 x 10⁴ cells/ml.  Mix 2ml of diluted cells + 2ml of the 1:1 mix of agar/2x medium and plate 2ml onto two dishes containing the previously plated 0.625% agar. (2.5 x 10⁴ cells planted)

Further dilute the above cell suspension [1ml cells + 3.0 ml medium] = 5 x 10³ cells/ml — Mix 2ml of this dilution + 2ml of the 1:1 agar mix and plate 2ml onto two dishes containing the 0.6% agar. (5 x 10³ cells planted)

What do you observe in the agar following one week’s incubation? What can you conclude from your own data plus those from your colleagues in the other "labs"?