I have attempted two clonogenic assays on MDA-MB-231 cells with a control and two knockdowns.
One group went into hypoxia for 72 hours and the other stayed in normoxia (both were in serum free media for this period only insulin and empty media). 500 cells were plated in each 10 cm dish and were grown for 20 days. There are barely any colonies in two dishes and too many in the others.
Can anyone suggest alternatives? I think plating was the problem here. I'm trying to look at the knockdown's ability to form colonies in hypoxia vs normoxia.
Joseph,
A few things to consider:
Joseph,
A few things to consider:
Seed 1000 cells in 6 well plate in serum containing medium. Don't use serum free medium. You dont have to wait for 20 days. You can stain the cells after 10 days.
it's better to perform the assay in 6-well plates. 500 cells should be good enough and seed them in medium with serum, later (once attached) you can change it according to your testing condition. Transfer them to hypoxia only after you have confirmed that the cells were attached, usually for 231 o/n is enough. Also, to get a better hypoxia effect, keep some media in the hypoxia chamber for more than 6hrs (depending on the volume) and use it to change the media next day for the set going inside the hypoxia chamber (Not sure if you have the glove box). Number of days may vary depending on the cell line and condition, therefore, best is to check regularly under the microscope and you can stain them the day you see the colonies.
We undertook clonogenic assay with these cells in serum containing medium ( as they dont like starvation) and as a point of note these cells were not great for clonogenic assay but we persevered. You should do a plating efficiency assay first with no treatment using variable cell numbers. For example plate 3 wells with 500 cells each, 3 with 1000, 3 with 1500. To get good data for hypoxia studies where you may see only small differences you need to have a good number of colonies in your control plate for example around 150- 200 colonies in your control is good to be able to pick up differences in treatment groups.
The issue you describe sounds like a pipetting/counting error. We use a haemocytometer for all our counting for clonogenic assay now rather than an autimated counter. Its very important that you disaggregate your cells properly by taking up and down through a pink or green needle directly before counting- i.e. if you have taken cells off with trypsin then added medium to denature and left them in a universal for a time before counting this will give you an off count. Thus take 1 sample at a time, take through a needle- count immediately- using the smallest gillson possible take out the required number of cells - add to plate- mix tube by inverting a few times before taking the next replicate and again before the third. These cells are " stciky" so its important to count and plate each sample one at a time.
If you have any issues pop me an email and i can send you a step by step protocol if its of any use. We have been doing clonogenic assay for many years and have seen lots of issues with different cell lines. The protocol we have established seems to work for most now but doing the plating efficiency assay first and making sure you get clean replicated without treatment is definitely worth doing with each new cell line.
If you still have reproducibility issues( which we still have with this line- and actually dont use it any more) I can also suggest an alternate assay- ( soft agar clonogenic or long term MTT - which have the same benefits as clonogenic assay)
Hope this helps :-)
We generally used 500-1000 cells in a 6 well format and grow them in complete media with changing old media with fresh one at every 48 hrs interval.
The mean plating efficiency was >60% at 2 weeks after plating when cultured in RPMI1640 containing 10% fetal bovine serum.
Hello everyone!
I plated 100, 200 and 400 cells (MDA-MB-231) per 12-well-plate (checking also in microscope), left it overnight and then 48hr treatment. 6 days later, I can barely see some individual cells in each well, including control. I don't think cell number plating is insufficent; indeed, other works mention even lower cell number...During clonogenic assay, is it suggested to change medium every 48-72hr or just leave the cells in incubator for 7-14d? Any other suggestions?
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