I did the MTT assay twice. The first time, the absorbance values was inconsistent among the wells. The second time, the values was somehow close to each other, but still different. Do you have any useful recommendations?
Thanks
Read until the cells without treatment get 1.0 absorbance
Hey Alaa A. Rashwan , I think u should think about the following:
1. Cell singularity: make sure when u trypsinize the cells, they become single. and if u centrifuge afterwards, make sure when u reconstitute in media, u pipette properly to ensure the cells are single again. Having cell clumps (>3 cells) might cause this difference
- P.S. avoid aggressive pipetting as it might shear the cell membrane and decrease cell viability
2. Cell Seeding: make sure u seed the same number of cells in each well. i.e. u have proper pipetting techniques. aspirating extra or less volume might contribute to such differences.
3. Cell Detachment: when u add drugs/PBS/MTT to the wells, make sure the pipette touches the wall and don't release the liquid in the center of the well as this might wash away some cells.
4. Media aspiration: if u r using an aspirator to remove the media, make sure u don't aspirate the cells as well by doing the following:
a. decrease the pump pressure to be the least.
b. don't touch the bottom of the well. this would increase the chance of cell aspiration
c. monitor ur aspiration technique by checking the wells before and after the aspiration
Hope this helps.