Hi,
So I have been using cell titer glo 2 in the lab, but we are getting some strange results for some reason. I did some troubleshooting and found gradients across the microplate as shown below. All the wells shown here have identical contents.
This can't be due to the edge plate effect from evaporation as when running experiments with cells the outer wells are not used and are instead filled with media (but no cells). I have quantified evaporation across the plate, and it is negligible.
It also is unlikely to be a pipetting error as it corresponds to too large differences and the pipetting was done with freshly calibrated pipettes and with particular attention to good technique.
I thought one possible explanation might be the focal height (we use autofocus). I thought perhaps if the detector was too high it might be reading spillover from several wells. I ran an experiment where I filled the wells with an ATP standard and ran the luminescence assay at different focal heights. There was no discernible change in the overall pattern at different focal heights.
I thought maybe the well positioning might be off but I checked and the correct plate dimensions appear to have been selected in all experiments plus we do not see the same pattern with fluorescence readings. The platereader also has an aperture which prevents spillover while reading luminescence values and this was fitted on all occasions. This trend also remains with two different plate designs and all the plates we use for these assays have white sides so direct spillover should not be occurring. Also, all the plates are read with the lid off so any scratches/inconsistencies/marks on the lid would not affect the results.
There is also no reason to think that there is photobleaching etc. going on from the measurement as when the same plate is repeatedly measured the values in each well remain relatively constant. The temperature was also controlled and kept constant at 25C
I am a little unsure what to try/how to troubleshoot further. I may try turning the plate around and seeing if the trend persists. Possibly I might also try re-analysing the data by looking only at the centre well results (we use a 3 x 3 matrix scan) or run an experiment measuring only in the well centre. I have contacted the manufacturer as well.
Is there anything obvious I could be missing here?
I think some simple controls would help you narrow the possibilities so you can narrow it down to 1) problem with the machine 2) problem with the plate itself or 3) problem with the samples. Flipping the plate around is a good one. Read it both ways rapidly and see whether the different cells go with the plate or the reader.
You might also wash the same plate and refill again with the same reagents to see if there might be some systemic issue with the way the wells are filled. You might also try the same reaction in a new plate.
These controls should help you narrow down the issue.
Dear Michael,
Thank you! I definitely will try reading in both directions quickly to see if that has an effect. I might also try repeating it with a single channel pipette in case there is an issue with the pipette itself.
What is the reading duration?
When the reading it too long there is a fading of the signal across the plate
Oded Babai, good question it is around 4 minutes. Unfortunately, I think that is not the case since the signal does not fade in wells after repeated reading. If we were observing fading signal, the values in each well should drop considerably after each reading (I repeatedly read for around 30 minutes). I would also have expected values to be strongest towards the top corners and weakest at the bottom corners, whereas what we observe here is that the values are strongest in the centre at the top and weakest at the centre bottom
I may try and repeat it with a single point rather than 3x3 matrix (as this will speed it up)
Can you dilute the sample in order to get lower signals, are the reading you show it ok or they are on saturation level?
Let me understand this problem correctly.
1) You seed the whole plate with the same cell density.
2) Dispensed by multichannel either 8 or 12.
3) Then you introduce CT-Glo to each well incubate at 10 mins.
4) Read with microplate reader
I have been doing readings with the Automation from Automated Liquid Handling system and may conclude few observations (Just Guessing)
1) Suspension cell behave differently in each well. They can proliferate at different speed but not very distinct.
2) Luminescence signal can deteriorate overtime. Not all the well but some of the well.
3) I normally use ATP to check for Automation Accuracy, at least something that i can control.
BTW signal over a million is too strong for your assay. You may want to consider reducing your cell density.
I am using cell titer glo as well to measure viability, but I couldn't get sufficient information about the filter and gain settings. How do you arrange your gain in the readings of CTG? And do you use filters or luminescence fibers? If you you filters, which one are you using?
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