Anchorage Independence Assay using Methylcellulose / Polyhema

 

Materials: Methyl Cellulose (Fisher, Cat #M-352)

Polyhema (Sigma, Cat #P3932)

 

        I.      Polyhema coating procedure (10X solution =120 mg/ml)

1.     Dissolve 2.4 g in 20 ml 95% ETOH by rotating ~ 8hr @ 65 C

 

2. Dilute 1:10 in 95% ETOH (final conc. = 12mg/ml). Coat plates with

4ml/100 mm dish

1.5 ml/ 60mm

0.5 ml/ well /6 well plate

Final density ~ 0.8mg /cm

 

3. Dry overnight @ 37C. Plates can be stored indefinitely prior to use.

 

 

 

      II.      Preparation of methycellulose

 

1.     Weigh out the required amount of methocel (1.5g/100 ml of media) into a glass bottle with a stirring bar inside (expect a 20-30% loss of methocel through the preparation process).

 

2.     Autoclave the flask with cap loose for 20 min.; dry for 15 min.

 

 

3.     Warm the correct amount of the right kind if media in the water bath (37C) for about 15-30 min. or until the entire bottle of media is warmed.

 

4.     Pour warm media into the sterilized methocel bottle. Shake well intil all methocel clumps are dissolved and the media begins to look like a milkshake.

 

5.     Stir the dissolved (non-clear) methocel at 4C overnight on a stirrer.

 

6.     Clear the methocel in a sterile 250 ml centrifuge bottle. Balance and centrifuge at 7,000 rpm for 30 min. to separate out methocel junk. Pour into a T75 flask and check sterility.

 

Use as soon as possible since the ingredients in the media tend to degenerate even at 4C. Generally, prepare the methocel one or two days before the experiment and use the same batch for the entire experiment until colonies are developed and counted (about 3-4 weeks).

 

    III.      Procedure

 

1.     Trypsinize cells. (Make sure cells are not growing too heavy, and do not let cells sit in trypsin too long, as they tend to clump up).

 

2.     Bring up in a few ml of media (approx. 4-5 ml dish/ 60mm, 1ml/ 35mm dish, 8-10 ml/ T75 flask).

 

3.     Count cells.

 

4.     Dilute cells to 105 cells/ ml with media.

 

5.     Triturate cells approximately 10 times up and down with a 1ml pipette to break up any clumps. Ascertain that all cells are single cells before introducing into methocel. If cells still clump after trituration more than 10 times, leave as is, and note the number of cell clumps/plate after seeding in plates.

 

6.     Add 2 ml cells to 8ml methocel in a 50ml tube precooled in an ice bucket.

 

7.     Pump the methocel up and down but not out (to avoid bubbles) in a 10 ml pipette.

 

8.     Seed 105 cells/5 ml /dish.

 

9.     Score bottom of dishes to check for clumps; mark the number of clumps observed under the microscope.

 

10. Feed dishes once/week with 1 ml methocel/dish.

 

11. Count colonies after 3-4 weeks (can use MTT to help visualize).