Essentially, the assay is same for suspension cells. The main thing is to dilute cells after treatment for clonogenic assay in a way that each well in the cell culture plate in which cells are to be grown in suspension receives a single cell. Therefore, dilution needs to be optimized and cells need to be suspended singly (without any cell clumps) and counted accurately to be able to seed in wells singly. For cells to give rise a cell population, the growth conditions (medium, growth factors, and CO2 levels) need to be optimum. It is a challenge for most suspension cells to proliferate initially (when present at very low density). 

It possible to use a semi-solid medium for cell cultures with methyl cellulose. So assay will be very similar to commercial MethoCult for haemopoetic CFU assay.

Hi Alexis ,

I'm trying lately to do CFA(colony forming assay) for LL2 cell line ( lewis lung carcinoma).

they are loosely adherent and floating cells.

usually in cells like these we make the CFA in semi solid medium like soft agar

but i face many issues : changing the medium each 3 days harm the gel , after 7 days when i want to stain with the crystal violet i find the gel close to liquid not coherent like the gel .

also, there is no occurrence in the number of cells i want to seed( i want to seed 1000 , but in most cases i don't get the number i want, in addition some cells make clump ) .

is there a better way than soft agar to make a CFA for these cells?

if not any advices or protocols may help me in this assay ?

i use in 6 well plate :

1% high gelling Agarose ( 1.5 ml )

2% low gelling agarose with medium (1:3.3 ) mixed with the cell suspension 1:1 . (1.5 ml )

then( 1 ml) medium .

thank you in advance .

try semi- solid agar medium or soft agar colony formation assay