We are trying to do soft agar assay in BT474 (luminal B) breast cancer cells.
I tried couple of times, but could not get even a single colony. Last time I even tried using MCF10A-Ras cells but could not get it work.
My protocol is I diluted 10X RPMI (Without folic acid, NaHCo3 and L-Glut) to 2X in milli Q water and added, 20% FBS, folic acid and NaHCO3 but did not add folic acid.
After adding equal volumes of 1% Agrose and 2X RPMI, I added 1.5ml/well in six well plate to make base layer, and kept the plate within hood for at least half hour. The same way I made top layer containing 10*4 cells/well, 0.35% agarose (final conc.). After 15 minutes the plate was put into the incubator. The next day I added 1ml/well media.
I can see the healthy cells under micorcope, but they do not divide at all.
The agarose I used is from sigma gelling temp of about 37
http://www.sigmaaldrich.com/catalog/product/sigma/a9539?lang=en®ion=AU
My question is could this be because of the unavailability of folic acid or agarose or some other reason?
I don't have experience with your cell line but I have used Noble Agar for my assays success. You may need to optimize the percentage of top agar for your cell line. Also cool your agar to 40C and warm your FBS media to 40C right before you mix and pour, to prevent growth factor degradation.
http://www.nature.com/protocolexchange/protocols/2611#/reagents
I have used low melting agarose from sigma for soft agar assays with colon cancer cells and MCF7. As Richard suggested, your agarose solution should be well dissolved and cooled to 40 degs before adding complete media to it. Your cells may get seriously affected due to heat stress if the top agar is not cooled to 40 degs.
Hi Abdul, we use 2 ml/well with 0.75% Agarose (final concentration) for basal layer and 1.1 ml/well with 0.4% Agarose (final concentration) as top layer. We add only 250 µl of fresh growth medium after 4 and 7 days. It is very important to add L-Glutamine to the medium. We used 200-400.000 BT-474 cells per well and got nice colonies after 12 days. In my experience, it is also important to use cells from a good growing cell culture. BT-474 are known to divide very slowly in 2D with a doubling time of about 7 days. As culture medium in 2D and in the soft agar assay we use RPMI with glutamax, 10% FCS and 10 µg/ml Insulin.
Hi,
We used soft agar colone culture for many cancer cell line in vitro. You need to use low temperature agar and adding red blood lysis from nude rat or nude mice. See "Bone Marrow Transplant. 1992 May;9(5):319-23. "An effective immunomagnetic method for bone marrow purging in T cell malignancies." by Wang et al.
Good luck with you EXP.
All good advice, but some cells like B cells get their receptors crosslinked and don't divide. There's an alternate assay using microscope slides and CO2 independent media. See Godar 1999 JID
Thanks your very much for everyone.
@Daniel Feger. So you seed 200-400 or 4x105 cells?
Definitely we are adding L-glut, but I forget to add folic acid, does it matter???
Also does it matter to use agarose or low melting agarose.
Hi Abdul,
we seeded 2x105 - 4x105 cells per 6well. Our medium contains folic acid per se. According the manufacturer the concentration of folic acid is 1 mg/ml. You should definitely use low melting agarose.
@Daniel Feger.
Thank you very much for your reply.
I am little concern about the number of cells as to me it seems too many number of cells per well?
Another question why do you use insulin in your culture as, if I am not wrong, it is not recommended by ATCC?
The agarose I used from sigma has gelling temp of 37, but we also have low melting agarose from biorad.
I would recommend to test different cell numbers: 0.5 - 4 x105 cells/well.
ATCC recommends Hybri-Care medium. This medium contains insulin (see medium formulation at ATCC website).
I think both agaroses should work.
Good luck with your experiments!
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