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Toxicity assays - Viability assays

Q: What kind of assay is Calcein-AM?

It is a non fluorescent, cel permeable compound. The cell needs esterasen to change the calcein-AM into calcein => this is fluorescent . (Esterasen zijn enzymen die esters splitsen in een alcohol en een zuur door middel van een reactie met water (hydrolyse).) Calcein blijft alleen in cellen met intacte membranen. => dead cells do not have esterasen

12 important questions on Toxicity assays - Viability assays

What kind of assay is the MTT assay? Name the enzymes

Only living cells can transform MTT into formazan = PURPLE
You need NAD(P)H dependent oxidoreductase enzymes to transform it.

1. NADH-dehydrogenase

2. Lactaatdehydrogenase

NADH wordt omgezet in NAD+

Made by mitochondria but NOT ONLY.

MTT creates formazan cristals => these are insoluble => you cannot measure with a Spectro photo measurement. => you need an extra step => you make it soluble with DMSO.

Are there similar assays that do not need the extra step with DMSO?

Yes -> you can use other assays that do not create these insoluble critalsXTT, MTS, WST => these use PMS (intermediate electron acceptor, 1-methoxy phenazine methosulfate) => this helps to carry on the electrons form the enzymes to the tetrazolium colour.

For how long should you incubate the cells with MTT?

2-4 hours

So, you will receive more colour (purple) when there is enough NAD(P)H. Where should you be cautious of?

ð Certain compounds can amplifiy the assay => by impacting the redox states => this does not mean that more cells are viable.

How does a Resazurin assay work? And wherefore do you use it?

It detect metabolic activity.

Resazurin enters both dead and living cells because it is cells permeable.

Resazurin will be => resorufin
It goes from blue to pink!
It also needs needs NADPH enzymes.
It also needs a redox reaction.
It is non-toxic

Do you also need the solubilisation like with the MTT assay?

No.

What are advances of resazurin?

Cheap.
Can be repeated on the same cell multiple times.

What kind of assay is neutral red? What happens in the cell?

Viability assay.

Accumulates in the lysosomes => vesicles in the cell containing enzymes; to break down old bacteria and waste products in the cell
Neutral red is water soluble.
Neutral red is a base (cationic), in neutral surroundings it is unloaded => diffuse through the membrane
When it comes into the cell it gets loaded and is not able to leave the lysosome => accumulation

=> Dead cells loose their pH-gradient => no ion trapping => no accumulation

What is an advantage of neutral red? What is a disadvantage?

96 well based
Cheap and fast = 2-3 h

Disadvantage: Caveat: Can be affected by compounds acting directly on lysosomes. e.g. chloroquine

What kind of assay is Calcein-AM?

It is a non fluorescent, cel permeable compound.
The cell needs esterasen to change the calcein-AM into calcein => this is fluorescent.
(Esterasen zijn enzymen die esters splitsen in een alcohol en een zuur door middel van een reactie met water (hydrolyse).)

Calcein blijft alleen in cellen met intacte membranen.

=> dead cells do not have esterasen

What can you use in the calcein-AM assay?

P-glycoproteïne (Pgp) inhibitor => because P-glycoproteïne (Pgp) pumps the calcein out of the cell.

What is an other fluorescence assay?

ATP assay.

Living cells have high amounts of ATP
ATP is quickly degraded by ATPases
Luciferin-lufierase + ATP emits photons (light)

Luciferin-based assays rely on the bioluminescent reaction catalyzed by the enzyme luciferase, which uses ATP as a cofactor to convert luciferin into oxyluciferin, producing light. The intensity of this light is directly proportional to the amount of ATP present, which reflects the number of metabolically active (viable) cells.

The question on the page originate from the summary of the following study material:

Toxicologie

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