Hi,
I am performing an MTT assay on HL60 and OCI AML2 cell lines,to detect the IC50 values for cytarabine. I needed to find the appropriate cell seeding density for which i initially seeded 0.1 million cells and later performed a double dilution to bring down the total cells per well to 50000, 25000, 12500, 6250 and 3125 cells. I set up 2 96 well plates to check the 0hour and 48 hr cell number. Literature mentions the doubling time of HL60 to be upto 48 hours and that of OCI AML2 to be 30 to 50 hours. Hence i kept my experiment time point upto 48 hours. for my 48 hour readouts, after adding MTT i could see formation of crystals in 3 hours yet i continued incubating upto 4 hours as per the standard protocol. However, on adding DMSO I found the crystals not dissolving to which i also tried pipeting the solution. The amount of MTT reagent added was 20ul for 200ul well volume and DMSO added was 100ul.
I am repeatedly facing the same issue where in some wells the crystals seem to dissolve completely while in some crystals stay in the solution even after placing on shaker for the estimated 2hr duration.
Please let me know where I am going wrong and also suggest an appropriate solubilizing agent.
Hi Manasi,
The formazan crystals formed after adding MTT (4 hour incubation) should ideally dissolve in 100% DMSO rapidly (Maximum 5 minutes should be enough to get a homogenous purple solution). To facililitate the mixing, we do gentle taping. We use MTT at 1 mg/ml final concentration and incubate for 4 hours, but never faced a situation where the crystals do not get solubilized in DMSO.
Ms. Manasi,
For clarification only, did you remove your conditioned media (used during treatment period) from each well before adding 100uL DMSO to each well or directly added DMSO (to the wells with cells+ media).
If you removed the media before adding DMSO, crystal will dissolve easily.
or If media is not removed, then it may not dissolve completely as your facing the issue.
Considering other option, if you have removed media, then added 100uL DMSO, try to increase the volume of DMSO to 200uL to all the wells or check and change the DMSO stock of yours.
Palaniswamy Ramaswamy
Yes I did remove the conditioned medium before adding DMSO. Mine being a non adherent cell line, I cannot entirely remove the media and keep around 10 to 20 microlitre in the wells and then add DMSO.
As per your suggestion I wil try to follow.
Thank you for your suggestions
- Badges
- Science topic