Cytotoxicity and Cell Viability Assays

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Questions and Answers

What does the variable IC50 represent in the provided model?

The slope of the response curve

The concentration of the tested inhibitor

The maximum value of the tested characteristic

The lethal or inhibitory dose sought (correct)

Which of the following best describes the relationship indicated by the slope 's' in the model?

The slope is independent of the inhibitor concentration

A higher slope indicates a lesser effect of the inhibitor

A flatter slope implies a nonlinear relationship of concentration and effect

A steeper slope corresponds to greater inhibitor influence (correct)

In the context of the equation, what are Ymax and Ymin used to represent?

The expected levels of IC10 and IC90

The specific doses of the inhibitor used in testing

The calculated maximum and minimum concentrations for Y

The overall limits of the response characteristic being observed (correct)

The notation [C] in the equation represents what?

The concentration of the current tested inhibitor Signup and view all the answers

What does a higher value of IC50 indicate about the inhibitor's effectiveness?

The need for a higher concentration for a 50% effect Signup and view all the answers

What is the purpose of incubating the plate in the dark after adding the MTT solution?

To allow living cells to reduce MTT to formazan crystals Signup and view all the answers

Which of the following is NOT a step in the MTT test protocol for cytotoxicity analysis?

Incubate with MTT for more than 4 hours Signup and view all the answers

What is the relationship used to determine the percentage inhibition of cell growth?

Absorbance of control - absorbance of sample x 100% Signup and view all the answers

Why is isopropanol or DMSO used after the MTT incubation?

To dissolve the formazan crystals into a purple solution Signup and view all the answers

What is the purpose of measuring absorbance at 570 nm and 690 nm?

To correct for background absorbance during the measurement Signup and view all the answers

Which method is used to assess cellular metabolic activity in relation to cell viability?

MTT assay Signup and view all the answers

What is a common mechanism by which physical and chemical agents can induce cell toxicity?

Destruction of cell membranes Signup and view all the answers

Which of the following parameters can be assessed using in vitro cytotoxicity tests?

Cell membrane permeability Signup and view all the answers

What is the primary purpose of using cell and tissue culture models in toxicity testing?

To provide rapid and cost-effective screening methods Signup and view all the answers

Which component of the MTT assay indicates viable cells after the assay procedure?

Purple formazan crystals Signup and view all the answers

What does an increase in cell adherence indicate in a cytotoxicity assay?

Cell growth and proliferation Signup and view all the answers

Which of the following factors is NOT assessed when determining cytotoxic doses in cell viability assays?

Thermal stability of the compound Signup and view all the answers

What is one of the key advantages of using in vitro assays over animal models in cytotoxicity testing?

In vitro assays allow testing of significant sample sizes rapidly Signup and view all the answers

What is the primary purpose of solubilization solutions like DMSO or isopropanol in the MTT assay?

To dissolve insoluble formazan crystals for quantification Signup and view all the answers

During the MTT assay, what parameter is measured to assess cell viability?

The absorbance of the colored solution at 500-600 nanometers Signup and view all the answers

What is the purpose of including control wells in the MTT assay?

To provide a basis for background correction Signup and view all the answers

Which of the following statements regarding resveratrol is correct?

It exhibits anti-inflammatory and antioxidant properties. Signup and view all the answers

What is the optimal incubation temperature for HeLa cells during the MTT assay?

37°C Signup and view all the answers

What is the importance of varying concentrations of resveratrol in the MTT assay?

To determine the threshold for cytotoxic effects Signup and view all the answers

What role do formazan crystals play in the MTT assay?

They are a resulting indicator of cell metabolism Signup and view all the answers

Which of the following best describes resveratrol's classification?

It is a polyphenolic compound. Signup and view all the answers

What is the purpose of the dose-response curve in the context of cytotoxicity assessment?

To evaluate the relationship between drug concentration and cell viability. Signup and view all the answers

What does the term LogIC50 refer to in this context?

The concentration at which 50% of maximal inhibition is observed. Signup and view all the answers

How is mitochondrial activity indicated in this model?

By recording changes in absorbance at specific wavelengths. Signup and view all the answers

Which mathematical expression correctly represents the model for calculating cell viability?

Y = Ymin + (Ymax - Ymin) / (1 + 10^((LogIC50 - X) * Slope)) Signup and view all the answers

What is indicated by the variable 'Ymax' in the mathematical model?

The maximum possible cell viability in the experiment. Signup and view all the answers

In a dose-response curve, what does an increase in slope indicate?

An increase in toxicity of the compound tested. Signup and view all the answers

What could be inferred if Ymin is significantly lower than expected?

There may be a cytotoxic effect occurring at low concentrations. Signup and view all the answers

What does the variable 'S' represent in the data provided?

The slope of the dose-response curve. Signup and view all the answers

What concentration of resveratrol corresponds to a Log10[C] value of -1?

$0.1$ M Signup and view all the answers

How is the IC50 value related to the effectiveness of a compound?

A lower IC50 indicates higher potency and effectiveness of the compound. Signup and view all the answers

Which of the following implications can be drawn from the experimental data at higher resveratrol concentrations?

There is a potential reduction in mitochondrial activity. Signup and view all the answers

What does a negative value of the slope indicate in this graphical representation?

Inhibition of cell growth increases with concentration. Signup and view all the answers

Which experimental measure is used to determine cytotoxicity in this scenario?

MTT assay which assesses cell viability. Signup and view all the answers

What does the variable 'F.straty' refer to in the context of the data?

The rate of cell loss due to treatment. Signup and view all the answers

Study Notes

Industrial Applications of Animal Cell and Tissue Cultures

Animal cells and tissue cultures are used in various industrial applications, specifically in cytotoxicity tests.
These tests determine the cytotoxic doses of different chemicals and agents.

Cytotoxicity

Cell health is assessed by viability levels and proliferation rates.
Physical and chemical agents can affect cell health and metabolism, leading to toxicity.
Mechanisms of toxicity include damage to cell membranes, disrupted protein synthesis, binding to receptors, inhibition of DNA synthesis, and enzymatic reactions.
Reliable and inexpensive short-term cytotoxicity and cell viability assays are crucial for determining cell death.

In Vitro Cell Viability and Cytotoxicity Assays

In vitro assays using cultured cells are used for chemical and drug screening.
Such assays are increasingly important in oncological research for evaluating compound toxicity and tumor growth inhibition during drug development.
These assays are rapid, inexpensive, and do not require the use of animals.
They are useful for testing many samples, and the assays are based on various cell functions.

Cell and Tissue Model System for Toxicity Testing

Preliminary steps include: cell/tissue culture model preparation, determining dose ranges of the substance, defining exposure time to the substance, and selecting the appropriate assessment method (cell viability, metabolic activity, proliferation rate).

MTT Assay

The MTT assay measures cellular metabolic activity, used as an indicator of cell viability, proliferation, and cytotoxicity.
A colorimetric assay reduces yellow tetrazolium salt (MTT) to purple formazan crystals.
Viable cells containing NAD(P)H-dependent oxidoreductase enzymes are responsible for the reduction of MTT to formazan.
Insoluble formazan can be dissolved and quantified by measuring absorbance.
The darker the solution, the more viable, metabolically active cells are present.

Determination of Resveratrol Cytotoxicity

Resveratrol is a polyphenol, a phytoalexin with a stilbene structure.
It's found in grapes, wine, cranberries, blueberries, and other herbs.
Resveratrol shows anti-inflammatory, antioxidant, cardioprotective, neuroprotective, and anticancer properties.

Procedure of MTT Assay

Materials: 96-well plate, cells, media, tested compound, MTT reagent, solubilization solution.
Steps: Cells seeding, Incubation with the compound/drug, Removing MTT and dissolving formazan crystals, Measuring absorbance.

Analysis of Cell Viability and Metabolic Activity by MTT Test

Steps involve removing media, adding MTT reagent, incubating, removing MTT, adding solubilization solution, mixing, measuring absorbance, and analyzing the results.
Analyze data to determine percentage inhibition of cell growth and plot dose response curve (to determine EC50 values).

Determination of Percentage Inhibition of Cell Growth

Determine the percentage inhibition of cell growth based on the absorbance of the control and sample wells.
Plot a graph showing inhibition of cell proliferation against the resveratrol concentration.

Dose Response Curve

A dose-response curve plots cell viability against different concentrations of a test compound.
EC10, EC50, and EC90 values on the curve represent the compound concentrations that affect 10%, 50%, and 90% of the cells, respectively. These are key for assessing cytotoxicity.

Mathematical Model

A mathematical model fits experimental data on cell activity over time to determine parameters like IC50 using models like Y=Ymin+(Ymax-Ymin)/(1+10^((LogIC50-X)*Slope)).

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Description

Explore the industrial applications of animal cell and tissue cultures, focusing on cytotoxicity testing. Learn how cell health is assessed, the mechanisms of toxicity, and the significance of in vitro assays in chemical and drug screening.