Soft agar-protocol - (Sep/15/2005 )

Hello
I just found this forum and I am very happy to see such a forum.

I am going to start a project with colon cancer cells that will be radiated. I would like to look at their colony forming abilities with a soft agar assay.

I have read some protocols, but I have not fopund any that show anything more than how to prepare the agar. Does anyone have a good protocol?
I feel really stupid, but I have questions such as
-does the cells go in between the agar layers?
-does one have to add extra medium during the growing time or will they survive with the nutrients and the moisture in the agar?

Thank you

SOFT-AGAR ASSAY

This protocol is designed such that from the same stock of cells also a proliferation curve can be initiated


- Experiment is performed on 6-well plates; 24.000 cells per well (although twice as many cells should be fine); perform in duplo.

- Prepare 3% agarose (Sigma type VII, cat# A-4018) in PBS
- Autoclave; store @ 4°C
- Just prior to use heat flask in micro-wave or boiling water; cool down to and keep
@ 39°C (keep sterile)

BOTTOM LAYER (1% )

- Dilute agarose 3-fold with 37°C complete media to get 1% suspension
- Use 2 ml per well
- Let cool down for 20’ RT


TOP LAYER (0,4 % )

- Prepare 0,8% agarose/media stock in (multiple) 50 ml tubes, keep @ 39°C; use for each in-duplo sample:

0,67 ml 3% agarose
1,83 ml complete media
2,5 ml 0.8% agarose/media mix

For each in-duplo sample add to this mix 60.000 cells (or more) in 2,5 ml media (24.000 cells/ml), to get 5 ml suspension

MIX THOROUGHLY (no air bubbles)

- Seed in each well 2 ml of this mix (equals 24.000 cells/well); in duplo
- Add water to empty wells/holes on plate
- Incubate 10’ @ 4°C or 30’ @ RT, then move to 37°C incubator
- Every 4 days add 3 drops of complete media(!)
- Count foci after 2(-3) weeks

thank you Theo22!

Anyone else?