Assays

Multiplexing assays

1. GF-AFC: fluorescence assay assessing live cells 2. bis-AAF-R110: cell- impermeant fluorogenic peptide substrate to measure the activity of a protease released from dead cells 3. Caspase-Glo 3/7 Assay: a luminescent assay that measures caspase-3 and -7 activities of cells, which are indicative of apoptosis.

Crystal violet assay

Assessing the quantity of attached bacteria in wells of a plate by applying and measuring a soluble stain bound to the bacteria

CyQUANT direct assay

Based on a cell-permeant DNA-binding dye in combination with a background suppression reagent that is impermeable to live cells. TheDNA-binding dye stains all cells, but in cells with compromised cell membranes the fluorescence is masked by the background suppressor Thus, the fluorescent signal is proportional to the number of live cells.

Annexin V assay

Ca2+ dependent phospholipid-binding protein that has a high affinity for phospholipid phosphatidylserineand binds to cells with exposed PS Labelled with a fluorescent tag Often used in conjunction with live/dead dye such as propidium iodide

ATP assay

Composed of: Detergent to lyse the cells ATPase inhibitors to stabilise the ATP that is released from the lysed cells Luciferin as a substrate Luciferase to catalyse the reaction that generates photons of light

DNA labelling assay - Label DNAs of adherent cells

Crystal violet assay Fluorescent conjugates

Membrane integrity assay - measurements of cell viability by detecting the membrane integrity of cells

Exclusion dyes Fluorescent dyes LDH leakage assay Annexin V assay

Protease viability marker assay

GF0AFC is a cell permeable fluorogenic protease substrate Aminopeptidase in the cell removes the Gly and Phe amino acids to release AFC and generate a fluorescent signal proportional to the number of viable cells

Trypan blue exclusion assay

Intact, functioning cell membranes exclude the trypan blue dye Compromised cell membranes cannot prevent trypan blue from entering the cell 'Dead' cells stain blue Cannot distinguish between apoptotic cells

MTT assay

MTT is reduced by active cells to insoluble formazan crystals (purple) w/ dehydrogenase enzymes Solubilize w/ detergent and quantify by spectrophotometry (OD) Use this as a comparison for levels of T cell proliferation compared to normal human controls to assess stage of disease progression in HIV/AIDS Results could be misleading as the tests agents could: - Reduce availability of MTT to the enzyme - Compete with MTT for enzyme binding site - Change the MTT reduction rate - Inhibit dissolution of formazan - Modify cell metabolism

Alamar blue reduction assay

Non-fluorescent dye with blue colour until it is irreversibly reduced to the pink colours and highly red fluorescent resorufin Used as an oxidation-reduction indicator in cells

Real-time viability assay

Real-time viability assay utilises an engineered luciferase derived form a marine shrimp and a small molecule pro-substrate Viable cells with an active metabolism reduce the pro-substrate into a substrate which diffuses into the medium where it is used by luciferase to generate a fluorescent signal

Why could MTT results be misleading?

Results could be misleading as the tests agents could: - Reduce availability of MTT to the enzyme - Compete with MTT for enzyme binding site - Change the MTT reduction rate - Inhibit dissolution of formazan - Modify cell metabolism

Functional assays - measure some aspect of general metabolism activity as a marker of viable cells

Tetrazolium reduction assay Alamar blue assay ATP assay Live cell protease assay Real-time viability assay

LDH leakage assay

When the cell membranes are compromised or damaged, lactate dehydrogenase, a soluble yet sable enzyme found inside every living cell, is released into the surrounding extracellular space