Hi everyone, I'm having some trouble with classical MTT assay. Me and my group have been doing MTT assay for a few weeks now, then suddenly it started giving us problem. I'm working in 24-well plate with BEK (Bovine Embryo Kidney) cells.
Cells are seeded in 1ml, I remove most of it, leaving 250 ul, before adding 40 ul MTT 5 mg/ml. After a few hours, I then add home-made lysis buffer (SDS 10% / HCl 0.01M) and finally incubate O/N at 37°C.
Formazan crystals are clearly visible on the bottom of the well before adding the lysis buffer, but then, the day after, all wells are completely yellow, except for a faint blue shade where crystals were more abundant, but not even comparable to deep blue wells I usually get.
So, crystals are made but just disappear after adding the lysis buffer. I noticed that this effect is already visible after half an hour: crystal completely deattach and form a sort of blue halo that quickly disappear after mixing the well, leaving it completely yellow, regardless of the amount of formazan.
What's going on there? I tried using a different MTT stock, but the outcome is always the same, so I reasoned that the problem might be the lysis buffer...
Instead of the last step and Instead of your lysis buffer, add apprioprate amount of pure DMSO, incubate at room temprature for 15 minutes. Then, read the absorbance.
Why are you using 24 well plates. You just need cell viability so go for 96 well plate. Secondly cell count should be at least 10X10^4 in each well. Your concentration of MTT is perfect. Here in 96 well plate only 10ul will be enough. Keep plates in incubator for at least 4 hours.
Now add 50ul DMSO in each well. Mix it well and take OD.
Try by this method. I hope you will get results for sure.
I agree with Arman Rahimi and Andleeb Khan. FIrst it is better to use a 96-well plate. Second, the lysis buffer is not required. After at least 4h incubation, add 50 or 100 ml DMSO, then read the absorbance. You can be sure from dissolving of the crystals by slow pipeting or shaking.
DMSO worked very well, thanks everyone for your help. One thing I'm not sure about: do you still read the plate at 570 nm, or does DMSO require a different wavelenght?
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