I have come across various methods and techniques.
Was wondering if people had better experiences or would prefer one method over another; advantages/disadvantages.
These particular experiments are on ovarian and some gastrointestinal cancer cell lines.
I am studying the effectiveness of specific agents in combination with chemotherapy for the survival and proliferation of these tumor cells.
Good Wishes.
I have used ATP lite (Perkin Elmer) to measure ATP in the cells. the higher the ATP the higher the cell proliferation.
In a classical assay, one assumes that a colony arises from a single cell. Therefore, one counts colonies or use other quantifiable methods, such as measuring an absorbance of a dye. See our paper below.
Link: http://www.ncbi.nlm.nih.gov/pubmed/12894224
In addition to the previous suggestions, Colony formation assay - you challenge single cells to form colonies in a normal 6-well culture plate - is a very easy and inexpensive technique. Count and plate ± 200 cells/well, keep them growing in the presence of your experimental conditions (chemo/control) and when you see colonies with the microscope (± 2 weeks), fix cells (PFA) and stain with crystal violet. Then count the colonies. You can use Image-j (or any other software) or do manually. If colonies are confluent, repeat with less cells. It is easy of you count and keep the cell/colony number around 200/plate in your controls. Counts >1000 may may result difficult to quantify (and confluence starts).
Alternatively, spheroid formation is also very nice and inexpensive, though a little bit more complicated. We use this with SKOV, and it works nicely. I can send you the protocol although it is quite standard and you'll find in pubmed (vinci et al, BMC biology 2012, 10:29 is one example).
Keep in mind, not all cell lines are able to form colonies or spheroids (reflect in fact to 'clonogenic' capability of cells), so you should test first their ability and in case adjust the cell number that you plate.
Still learning; that would be wonderful Andrea if you could forward me the protocol. How do you end up counting the cell lines then? Is it the size of the spheroids? Additionally, what about issues regarding contamination given longer periods of growths? How do you re-use the cells for IHC or PCR? Do you just trypsinize them?
The clonogenic assay evaluates cellular clonogenic potential (ability of a cell to form a colony). In this assay, clonogenic colonies (usually defined as colonies with 50 or more cells) are considered as survivors, based upon which the clonogenic survival can be measured. On the other hand, abortive colonies (usually defined as colonies with less than 50 cells) are considered not as "dead" cells but as "inactivated" cells.
There are various modes of such clonogenic inactivation including premature senescence. Depending on the methods employed, underlying modes of cell death or inactivation change: for instance, senescent cells are clonogenically inactive (evaluated as non-survivors with the clonogenic assay), but metabolically active (evaluated as viable cells e.g., with the MTT assay).
Therefore, the methods need to be selected depending on what you actually wish to look at.
An automated counting option may be helpful to you here. The GelCount for example can unbiasly count only spheroids you want (ie those over 100 um) and not count the rest. It can provide additional information like average colony diameter.
Here is a webinar on some of the merits of automated colony counting.
https://www.scintica.com/webinar-automated-colony-assay-counting-advancing-the-frontiers-of-cancer-research/
- Badges
- Science topic