Hello!

I am just here to ask for some insight about the MTT procedure and why my experiments may have failed. I am completing an honours year and would like any tips or tricks you may have to assist!

I am testing for neurotoxicity of various chemotherapy agents on primary cortical neurons obtained from sprague-dawley rats at embryonic day-18. The cortices are processed and plated in 96-well plates at a density of ~52,000 cells/well, experimentation begins on days in vitro 10-12. The culturing method consistently produces wells which maintain 97% post-mitotic neurons to ~3% astrocytes. I have previously performed successful MTS assays on this model, however, recent constraints required MTT-assay this time around. We used the Invitrogen CyQuant MTT kit, the MTT-solution was dissolved in 1mL of sterile PBS only 1-day before experiment. The SDS-HCl solution was prepared ~1 hour before its addition to wells.

The drug exposure prior to the MTT-assay went well, upon addition of the tetrazolium reagent, there was visible formazan crystal production within the wells after 4-hours of incubation (37C, 5% CO2). Prior to adding the MTT-reagent, Neurobasal media (2% B27, with Phenol Red) was aspirated and replaced with BSS at a pH of 7, this was done to remove the impact of phenol red on OD measurement at 570nm.

The SDS-HCl solution was added and was checked various times between 2-24 hours and no colour change has occured. The wells are currently in the incubator and will reach 48 hours of incubation in a few hours. Unfortunately I do not have a photo of the wells available currently, however, the solution in SOME wells appear heterogenous with a layer of oil? (I am not too sure), other wells have become cloudy and finally, some wells have lost media volume (I assume that excessive time in the incubator has evaporated some media). Immediately after the SDS-HCl solution was added, the wells were mixed by pipetting the solution in the wells and also using the shake feature of a Cytometer 5 imager (1 Min / Slow orbitals).

I am wondering if anyone on here is able to provide any information about why my experiment is not developing. I believe it may be linked to the SDS-HCl solubilisation step rather than the MTT-reagent (there was visible formazan crystal production). I appreciate any help someone may have!