L4 Introduction to end point measurements for toxicity testing in cultured cell systems Flashcards
To introduce the general principles of end point determination in toxicity testing and to discuss some of the key approaches used to study cell viability, proliferation and metabolic activity. Understanding of the basic requirements of end point measurements. Ability to discuss key methods used to monitor cell viability, proliferation and metabolic activity. Ability to explain how to combine them effectively in a tiered strategy of toxicity testing.
What is end point determination?
A measurement as a response to a toxic agent (or protective effect of a molecule against toxic agent).
What are the four major types of end point?
Cell viability.
Membrane leakage.
Cell growth/metabolic activity.
Cell morphology.
How is cell viability assessed as an end point?
Essentially measure of living and dead cells. Cells can be counted in a haemocytometer chamber but doesn’t distinguish. Dye exclusion and update can be used. Typan blue taken up by non-viable cells, and neutral red is taken up by viable cells.
What is the duel fluorescence assay in cell viability end point measuring?
Using two fluorescence probes which are taken up by either viable or non-viable cells.
e.g.
fluorescing dictate taken up by living cells, fluoresces green after cleavage by esterase’s.
pro podium iodide taken up by non-viable cells and enters nucleus, fluoresces red.
How is membrane leakage assessed in end point measurement?
Lactate dehydrogenase (LDH) released into culture medium when cell membranes become damaged. Their activity in culture medium is detected. Can be initiated by toxic effects of Cd2+ in assay
How is cell growth and metabolic activity assessed in end point measurement?
By using a cloning efficiency assay or measuring DNA synthesis changes.
DNA synthesis assay involves BrdU labelling, where cells go through replication process and labelled brdU incorporated into DNA for thymidine. Only proliferating cells are fluorescently labelled.
Can protein synthesis be used as an end point measurement?
Yes, either total protein measurement or specific protein changes can be used as end point measurement.
Can metabolic assays be used?
ATP levels can be assessed. MTT is also good assay for end point measuring. Reduced MTT activity indicates reduced cell viability.
What four components of a cell can be used for cell morphology end point measurement?
Changes in size, shape, membrane integrity (blebbing), cell organisation (in colonies, indicative of cell interactions and cell behaviour)
