protocol 10: MTT and LDH assay

we use MTT and LDH assay to do what

determine whether treatments induce apoptosis or necrosis in CHO cells

MTT assay determines

overall viability and cell death

LDH assay determines

breach in the integrity of plasma membrane releasing intracellular contents, necrosis then occurs

death with an intact plasma membrane is

death via apoptosis

MTT is converted to formazan by what

mitochondrial reductase

MTT assay is a direct and indirect measure of

direct: mitochondrial function

indirect: cell viability

chemical compounds interfering with MTT assay are

reducing compounds that lead to non-enzymatic reduction of MTT to formazan

what other chemical are known to interfere with MTT assay

the ones that uncouple electron transport from oxidative phosphorylation of ATP

treatments with MTT assay

ascorbic acid, vitamin A, sulfhydryl-containing compounds, and dithiothreitol

what treatment results in production of formazan and higher background absorbance

long term exposure of MTT assay reagent to light and elevated pH of culture medium

MTT reagent

exhibits cytotoxic effects, which increase over time

the rate of tetrazolium reduction reflects

general metabolic activity or the rate glycolytic NADH production

MTT reduction being observed is also due to

cytoplasm, membranes in endosome/lysosome compartnments, and plasma membrane

LDH assay provides a direct measurement of

LDH released by necrotic cells

lactate dehydrogenase (LDH)

converts lactate into pyruvate

reduces enzyme NAD+ to NADH

LDH assay electron

MPMS+ picks up it from NADH and is reduced to MPMS

MTT assay electron

MTT picks it from MPMS and is reduced

LDH is not released from

living or healthy cells

LDH is stable in

media after it release

LDH release is

quick

with interfering with the LDH assay, what increases of background absorbance in an assay

human and animal sera contain varying amounts of LDH

in cell-mediated cytotoxicity assays, what influence this assays results

the amount of LDH released from damaged effector cells

what else influence the LDH assay

substances which inhibit LDH or enzyme activity

Pyruvate

inhibitor of the LDH reaction

both MTT and LDH levels up results in

apoptosis

only LDH level up and MTT level down results in

necrosis