I'm looking to do a time course realtime-glo vitality assay on MDA-MB-231 in a 96-well plate. I plan on doing 4 different seeding concentrations to assess linearity of signal, however there is some conflicting information on both doubling time and initial seeding density for the vitality analysis which makes it difficult to calculate an accurate range.
1) They seem to grow very quickly, typically doubling every 24hrs, which is inconsistent with other sources at 35-40hrs. Maybe this has to do with my media preparation (DMEM, 10%fbs, 1% anti/anti)?
2) 1 is primarily important because my time course is every 24 hrs for either 3 - 4 days. Some references are seeding at 5k cells per well, however if my doubling time is 24hrs then I will reach 125% confluence on day 3. Now from other sources I see seeding densities at 100,500,1000,2000 cells/cm^2, this seems fairly low since even the highest seeding density would be less than 700 cells per well.
Suggestions? I was thinking 500, 1000, 1500, 2000 cells per well might be a reasonable trade off?
Also one more question, I see some inconsistencies in total volume per well. Typically I use 100ul of total volume in a 96-well, though I'm seeing in the protocol closer to 200ul. I know the wells can handle that volume, but I assume that would alter growth of the cells owing to less oxygen / c02 exchange. Any suggestions on that as well?
Hi Martin,
Your range seems fairly accurate. I usually plate 1000 cells and can go up to 6 days with that seeding density. A volume of 100ul is fine.
1) 24 hour doubling time is normal for MB231. If cells are happy with a high seed count, they go right into exponential, if they aren't as happy then they'll go to lag phase. For example if you spike frozen stock into a T75 then you'll see the 35-40 hour doubling time. Take a 90% confluent split and put it in a T75 you'll see 24 hour doubling.
2) I don't know the goal of your experiment, but the range seems fine. Don't forget to account for lag phase length in your calculations.
3) I use 100-150 uL per well. You are correct to worry about CO2 exchange, but also keep in mind the nutrient availability and space availability is just as important. Depending on experiment length you may need to do daily media exchanges.
Ok, that definitely explains the discrepancy. It just surprised me a bit because I wasn't expecting them to grow that fast.
I'm not sure we will be able to do daily media changes though. We are doing a time course vitality assay every 24hr for 4 days on multiple drug conditions and concentrations. Admittedly there will be some toxicity from nutrient-loss, waste, PH changes, etc, however I'm thinking we should be able to account for these effects with our controls. Otherwise we have to change media with new vitality-reagent and drug each day. It's doable certainly, but for a small scale I think it's ok to try out and then consider the amendment if the time-course fails.
Thanks Daniel
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