Hi all,
I have been doing drug screening on clinical samples and multiple 384-well plates are used for each samples. However, when we look at the cell number in the DMSO control on each plate, there is a trend of decreasing number of cells from plate 1 to plate 5. There is no problem within the plate i.e. cell numbers are even in the same plate.
The way I do it is to use a Pipetboy, mix the cell solution a few times, and take 8 mL and put it in the reservoir. Then they were pipetted them using a electronic multichannel pipette which can dispense 4 columns of wells for each aspiration. I rocked the reservoir back and forth before each aspiration. After finishing one plate, I mix the cell solution thoroughly again and take another 6-8 mL of cells to the reservoir. The tips and reservoir I used are both from Integra.
This happens to both cell lines and primary samples, and it has been bothering me for a long time. Has anyone seen the same problem and has a solution for it?
Many thanks.
When you say the cell number decreases from plate to plate, is it that the number of live cells decreases, or the total number of cells? Maybe the cells are dying due to the change in atmosphere (low CO2) affecting the pH of the medium, or to physical disruption by all the pipetting.
Hi Adam B Shapiro,
It can be either but the observation is based on the viable cell numbers after 4 days of culture. I saw the same thing using conventional Cell Titer glo reagent and cell counting using imaging-based profiling. The cells were left in the hood from the first to the last plate for less than 10 minutes when I was using the cell line. Probably much longer when I am using the clinical sample. I will try to test what you have suggested. Thanks.
Kent Fung Looks like a classic (non-representative) sampling issue to me. It may be difficult to cure due to the low volume of each sample. The fact that the count is falling suggests your removing larger numbers of cells at the start which on a number-basis are the smaller ones. Trying to change the position from which you sample or (better still) stirring the 'suspension' (magnetic stirrer) while removing the sample may work if the solution/suspension volume is large enough.
Hi Alan F Rawle That is also something I have considered. Unfortunately there isn't a magnetic stirrer (the apparatus) clean enough that I would put them in a TC hood. If I dispense them quick enough I might just get a larger reservoir and pull everything in, and rock the reservoir between each dispense.
Kent Fung I would speculate that rocking may actually be causing segregation in the sample between the larger or more dense cells. Other solution (not a bad pun!) is to integrate the values from all wells/plates and thus measure the entire 8 mL of sample. No sampling errors if all is measured...
@Alan F Rawle I decided to go for your suggestion using magnetic stirrer. If you still se this thread, can you share your view on using glassware (cleaned and autoclaved) that has been used for other chemicals? I assume jt should be fine but I am also worried that chemical might be carried over.
Kent Fung Nowhere in your description do you provide any quantitative information. All we have is a detailed methodology and 'there is a trend of decreasing number of cells from plate 1 to plate 5'... [The cell count could decrease from 5 to 4 or 5000 to 4995 based on this comment]. You do not say what tests the plates will be used for beyond 'drug screening'. So, I can only rely on guesswork and hunches. So, please provide quantitative data to disprove my thesis which is: your initial sample is contaminated with cell debris and other rubbish and all you have is segregation taking place loading more rubbish into the earlier wells and thus decreasing count as we progress. This is the most common observation I have in such systems used in the manner that you state. OK, what filter are you using to load the samples into the wells? With tiny sample amounts cleanliness and freedom from contamination is vital. Please disprove my thesis with data. Thank you.
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