Cell Bio and Genetics Lab- Procedure 8 MTT and LDH Assays

MTT assay

allows us to determine overall viability and cell death

LDH assay

determine whether there is a breach in the integrity of the plasma membrane

What is an indicative of necrosis?

breach of in the integrity of the plasma membrane

Death via apoptosis

death with an intact plasma membrane

What is MTT also called?

3-(4,50dimethylthiazol-2-yl)-2,5-duphenyltetrazolium bromide

What is MTT converted to?

Formazan (yellow to purple)

What converts MTT?

Mitochondrial reductase

What is known to interfere with the MTT assay?

Chemicals that uncouple electron transport from oxidative phosphorylation of ATP

What are some examples of chemicals that can interfere with MTT assay?

ascorbic acid, vitamin A, sulfhydryl-contain compounds (reduced glutathione, coenzyme A), and dithiothreitol (DTT)

What will happen if the MTT assay has long term exposure to light and has elevated pH of culture medium?

may result in production of formazan and higher background absorbance

What does the MTT assay have that increases with time?

cytotoxic effects

What does the rate of tetrazolium reflect?

general metabolic activity or the rate of glycolytic NADH production- generally due to mitochondrial activity

What other components contribute to MTT reduction?

cytoplasm, other associated membranes in the endosome/lysosome compartment, and plasma membrane

What changes can also cause MTT reduction?

change with culture conditions

What does the LDH provide a direct measurement of?

LDH released by necrotic cells

What are the colors when MTT is converted into MTT formazan?

(yellow to purple)

What converts Lactate to pyruvate?

LDH (lactate dehydrogenase)

What are some assumptions about LDH during experiments

• LDH is not released from living or healthy cells

• LDH is stable in media after its release, and LDH release is quick

What may influence assay results in cell-mediated cytotoxicity?

• the amount of LDH released from damaged effector cells

• substances which inhibit LDH or enzyme activity

Pyruvate

an inhibitor of the LDH reaction and is contained in some formulations of culture media

What is an alternative to harsh stop soultions?

Acidified isopropanol

What is a downside to isoproanol?

That its absorbance must be measured immediately since the color is not stable in isopropanol

How does apoptosis differ necrosis?

• Purpose

  • Apoptosis is a programmed process that removes unwanted or damaged cell

  • Necrosis is an accidental process that occurs in response to injury

• Process

  • In apoptosis, the cell’s contents break down and are packaged into membrane packets for immune cells to collect

  • In necrosis, the cell’s contents spill out and cause inflammation

Apoptosis can be triggered by which two pathways? Outline the major events of each pathway

• Mitochondrial pathway (Intrinsic) which is initiated by internal cellular stress signals

  • Internal stress signals

  • Formation of the Apoptosome

  • Activation of Caspase-9

  • Caspase Cascade

• Death Receptor Pathway is triggered by external signals via specific death receptors on the cell surface

  • External death signals

  • Formation of the DISC

  • Activation of Caspase-8

  • Caspase Cascade

  • Cross-talk with Intrinsic Pathway

How is proptosis triggered? Outline the major events of the pathway.

• Triggered by infection

  • Detection of danger

  • Inflammasome formation

  • Activation of caspase-1

  • Processing of cytokines

  • Gasdermin D cleavage

  • Cell Membrane Perforation

How is Necroptosis triggered? Outline the major events of the pathway

• Death Receptor Activation

  • Formation of the Complex I

  • Formation of the Complex II

  • Necrosome Formation

  • MLKL Activation and Translocation

  • Cell Death and Inflammation

What color change is expected for the MTT assay?

Yellow to Purple

What color change is expected for the LDH assay?

Yellow to Blue

How can MTT and LDH assays be used to determine cell viability?

• MTT assay by quantifying the amount of formazan produced by mitochondria in living cells as dead cells can’t do this

• LDH measures the amount of lactate dehydrogenase (LDH) enzyme in the extracellular medium to assess the cell membrane’s integrity

The MTT assay provides a/an _________ measure of cell viability while the LDH assay provides a/an ________measure of cell necrosis

indirect, direct

Lysate

a fluid that contains the contents of a cell that has been broken down or lysed

Why are protease inhibitors included as a component of lysis buffer?

to prevent the degradation of extracted proteins

To prepare samples for Western blot or various other analytical procedures, the cells must first be lysed. Explain how the structure of Triton X-100 enables cell lysis to occur upon the addition of lysis buffer.

disrupts the cell’s membrane lipid bilayer and will destroy the membrane structure thus releasing cellular components into the lysis buffer

You are performing a sensitive assay that requires a minimum of 10 ug of protein. Your lysate concentration is 250.5 ug/mL. Calculate the volume (in ul) of cell lysate required to obtain the 10 ug of protein you need to perform the experiment.

40 ul/ 39.92 ul

You have prepared three samples of lysate and the concentration of protein for each sample has been determined. You wished to compare the three samples with regard to the amount of a specific protein.

a. Explain why the volume of each sample is not a valid basis for comparison

b. What can be used to compare the samples and ensure that equivalent amounts of each of the three samples are being compared?

a. The volume of each sample is not a valid basis for comparison because the concentration of protein within each sample can vary meaning that even if the volumes are the same, the actual amount of protein present could be significantly different between samples

b. To compare the samples and ensure equivalent amounts of protein are being compared, calculate the volume of each sample needed to load a consistent amount of protein based on their respective protein concentrations. This can be achieved by using the following formula:

Volume to load = (Desired protein amount) / (Protein concentration of the sample)