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