There are several factors that could contribute to your intriguing observations. Firstly, the origin of your "NIH 3T3" cells. Because murine embryonic fibroblasts (MEFs) from certain strains of mice grown using the NIH's "3T3" protocol are sometimes referred as "NIH 3T3" cells. When these MEFs maintained in culture for a long time inappropriately, these cells can undergo transformation spontaneously and could give rise to colonies in soft-agar assays. Secondly, when excess number of NIH 3T3 cells are used in the assays, certain small colonies could appear after prolonged incubation of plates. Therefore, the cell number in the assay and the incubation time need to be considered using approipriate controls. In summary, you really need a negative and a positive control to compare your "NIH 3T3" cells in soft-agar assays.

Cell density I'm using is 2000 per well on 96 well plate. It is lowest of range suggested by protocol. Not transfected cells are my "negative" control. Cells transfected with plasmid expressing KRAS oncogen are positive control. So basically I'm curious if my "negative" shall have any colonies - from experience of someone who already used these cells in this assay. Today I started this assay with two cell lines - NIH 3T3 and cos1. I wish/expect I will have no colonies in not transfected cells (cell line only).

What is known about NIH 3T3 cells? NIH 3T3 is not a "cell line". These are primary MEFs that are maintained in culture using the "3T3" protocol. If it turns out that the "negative" control is forming colonies in your assay, these cells are not suitable (for the assay).

I think when most people refer to NIH 3T3 they are referring to an immortalised murine fibroblast line (not a primary line as Divaker alludes to above).

http://en.wikipedia.org/wiki/3T3_cells

http://www.atcc.org/Products/All/CRL-1658.aspx#generalinformation

It is possible that the particular cell line you are using has been over cultured and become even more "immortalised". What passage level were the cells that you used (if know)?

Another possibility (as mentioned by Andrzej above) is the density you are plating at.

The below protocol is from "Transformation Potential of Ras Isoforms Correlates with Activation of Phosphatidylinositol 3-Kinase but Not ERK*"

http://www.jbc.org/content/279/36/37398.full

"Soft agar assays were performed according to published methods (33). Stable clones of NIH3T3 cells were plated onto DMEM soft agar plates at a density of 1, 2, and 6 × 104 cells/plate (35-mm plate). Plates were incubated for 3 weeks and examined by microscopy"

I am assuming from your question that there was not a significantly greater number of colonies forming in you Ras transfection than in the neg control?

Another alternative is to get some murine primary fibroblasts and use these (Lonza and several other companies sell them).

I agree with Gary that most scientist, who are familiar with the original description of NIH 3T3 cells (included in the web link provided) are also aware that MEFs had undergone a "crisis" in culture upon a prolonged culturing (using the 3T3 protocol) and cells had undergone a spontaneous immortalization. Therefore, it is critical to know whether further culturing of these MEFs has resulted in a "transformed" phenotype (including the loss of contact inhibition, serum independence, and colonies in soft agar). If yes, these cells are not suitable for assay.

I'm afraid that conclusion is that these cells I'm using now are not suitable - or assay is unsuitable. I expected that not transformed cells will not form any colony. They did. yesterday I started new eperiment using cells I got from friendly lab as "NIH 3T3" cells. I'm using quotation marks "" because it looks like these cells lost requirement for contact to stay alive. As you mentioned they probably had undergone spontaneous immortalization (judging from former results). Yesterday I started new experiment - paralell with these "NIH 3T3" and with cos1 (these I got from ATCC). Transfected with LTK variants, KRAS, empty vector and not transfected cells. Just now watching them to see how experiment will develop. Basically I was curious if anyone can tell me that these cells - in this case NIH 3T3 and cos1 can (or can not) to grow on soft agar without any modifications.

The answer for the original NIH 3T3 cells that are described in the web link by Gary is NO (because the original cells were not transformed).