Cell Viability Flashcards
What is the role of mitochondrial dehydrogenase in WST-1 assay?
They cleave the WST-1 tetrazolium salt into a soluble formazan dye, reflecting metabolic activity and thus cell viability.
Why are triplicates used for each treatment condition in the WST-1 assay?
to ensure accuracy and improve data sets for statistical analysis.
What wavelength is used to measure WST-1 absorbance and why?
420-480nm to detect the absorbance of the formazan product, a referecne at 600nm corrects the background.
What is the function of media in the three empty cells?
They act as controls, used to measure the baseline absorbance from media.
How does triton-X 100 affect OAW42 cells in this experiment?
It disrupts cell membranes, leading to a loss of viability and a reduced WST-1 signal.
What is the main difference in how WST-1 vs. Trypan Blue assesses cell viability?
WST-1 measures metabolic activity, trypan blue assesses membrane intergrity.
What does a low absorbance reading indicate in the WST-1 assay?
Fewer metabolically active cells present in the well.
Why must the cell culture plate be tilted before removing media?
To avoid disturbing the adherent cells on the cover slip, which could affect staining and imaging.
What is the purpose of using Trypan Blue in this experiment?
It stains non-viable cells (with compromised membranes), while viable cells exclude the dye.
Why is methanol used as a treatment in both experiments?
It fixes and kills cells allowing comparison with other treatments to assess loss of viability.
Why are coverslips mounted upside down on onto fructose mountant?
To ensure that the stained cell surface is in contact with the microscope slide for optimal imaging.
What are the two functions of washing with the PBS before and after treatment?
To remove media that may interfere, and to reomove residual treatment chemicals afterward.
Why is only 150uL of methanol used, compared to 1ml of other treatments?
Methanol is most potent and does not require high volume.
What can be inferred if Triton X-100 treated cells appear dark blue under microscope?
Cell membranes have been compromised, allowing trypan blue entry - cells are not viable.
