Practical NBT assay AI generated

Question 1

Describe the NBT assay in the context of studying macrophages.

Answer 1

It is a method to measure the capacity of macrophages to produce oxygen radicals by using NBT solution and analyzing the color change to formazan.

Question 2

What is the role of macrophages in the immune system?

Answer 2

Macrophages belong to the innate immune system and have a general clearance function, often referred to as ‘scavenger macrophages’.

Question 3

How is the color reaction in the NBT assay measured?

Answer 3

The color reaction is measured in a spectrophotometer after the released solid formazan is dissolved by DMSO.

Question 4

Define the primary immune response initiation involving macrophages.

Answer 4

It includes phagocytosis of antigens, degradation of pathogens to antigens, and presentation of antigenic determinants to CD4+ Th cells.

Question 5

What are some functions of macrophages in the body?

Answer 5

Macrophages act as garbage collectors, removing around 10^11 red blood cells daily in humans and playing a crucial role in immune responses.

Question 6

Describe the role of monocyte-derived macrophages in immune responses.

Answer 6

Monocyte-derived macrophages play a crucial role in the initiation phase of immune responses.

Question 7

What is the function of Fc-receptors on macrophages’ surface?

Answer 7

Fc-receptors on macrophages’ surface allow them to efficiently clear immune complexes (antigen-antibody complexes).

Question 8

How do macrophages present antigenic determinants to T helper cells in lymphoid organs?

Answer 8

Macrophages present antigenic determinants via MHC class II molecules and interact with T helper cells in lymphoid organs like the spleen.

Question 9

Define phagosome and phagolysosome in the context of macrophages.

Answer 9

Phagosome is the membrane enveloping bacteria, while phagolysosome is where bacteria undergo degradation within macrophages.

Question 10

What are some characteristics of the endosomal compartment of macrophages?

Answer 10

The endosomal compartment of macrophages has a low pH, contains around 40 acid hydrolases, and can produce nitrogen and oxygen radicals to kill and degrade microbes.

Question 11

How do phagocytes generate an ‘oxidative burst’ upon stimulation?

Answer 11

Phagocytes generate an ‘oxidative burst’ by increasing O2 uptake, converting O2 into superoxide anion, and further converting it to H2O2, which exhibit microbicidal activity.

Question 12

Describe the process of culturing macrophages in an experiment.

Answer 12

In the experiment, macrophages adhere to the culture surface, are cultured overnight, and then washed for further analysis.

Question 13

Describe the importance washing cells in a cell culture assay.

Answer 13

Washing cells helps standardize culture conditions, ensuring cells start under comparable conditions and preventing unwanted reactions.

Question 14

How is the conversion of O2 to the superoxide anion measured in a cell culture assay?

Answer 14

It can be measured using Nitro-Blue-Tetrazolium, which is converted by the superoxide anion to a blue solid formazan, measurable with a spectrophotometer.

Question 15

Define positive and negative controls in a cell culture assay.

Answer 15

Positive control involves using a known stimulus to elicit a specific reaction, while negative control involves having no cells present.

Question 16

What is the purpose of examining the effect of different yeast concentrations in a cell culture assay?

Answer 16

To observe how macrophages phagocytose yeast and understand the impact of yeast concentration on the process.

Question 17

Describe the potential consequences of wells being left without medium for too long in a cell culture assay.

Answer 17

Leaving wells without medium for an extended period can cause stress to the cells.

Question 18

How does Nitroblue tetrazolium (NBT) contribute to a cell culture assay?

Answer 18

NBT diffuses into the cell and is converted by the superoxide anion to a blue solid formazan, aiding in measurement.

Question 19

What happens if all reagents are combined without washing cells in a cell culture assay?

Answer 19

The solution turns blue without the presence of macrophages at the end, affecting the accuracy of the assay.

Question 20

Describe the behavior of macrophages in a cell culture assay regarding the bottom of the plate.

Answer 20

Macrophages tend to stick to the bottom of the plate and form a cell layer, requiring careful handling to maintain the cell layer integrity.

Question 21

Describe the process of removing excess NBT from the culture in the given content.

Answer 21

Excess NBT is removed by adding methanol to fix the cells and stop the reaction, allowing the amount of formazan produced to be determined.

Question 22

What is the purpose of adding KOH in the content’s process?

Answer 22

KOH is added to permeate the cell membrane and allow DMSO to dissolve the blue formazan particles for coloration measurement.

Question 23

Define the role of a spectrophotometer in the context of the content.

Answer 23

A spectrophotometer, also known as an ‘ELISA-reader,’ measures color intensity using filters corresponding to specific wavelengths.

Question 24

How are the absorption results interpreted in the given content?

Answer 24

Absorption means in columns 3, 4, and 5 are similar, with columns 3 and 4 slightly higher than 5. A small absorbance in the negative control of column 2 is noted.

Question 25

Describe the conclusion drawn from the absorption results in the content.

Answer 25

The aim was to understand the production of oxygen radicals by activated macrophages. Column 4 and 5 show similar outcomes to column 3, with the highest color intensity in column 4 due to yeast concentration.

Question 26

What is the significance of the small absorbance in the negative control mentioned in the content?

Answer 26

The small absorbance is attributed to measurement errors, as the negative control lacked a stimulus, resulting in low color intensity.

Question 27

Explain the process of measuring color intensity using a spectrophotometer in the context of the content.

Answer 27

The spectrophotometer measures the intensity of color by using filters that correspond with specific wavelengths, allowing for quantitative analysis of the amount of formazan produced.

Question 28

Describe the purpose of resuspending the stimuli before adding them to the wells.

Answer 28

To mix the stimulants and distribute them homogenously within the wells to prevent yeast particles from sinking to the bottom.

Question 29

Define negative controls in this experiment and explain their significance.

Answer 29

Negative controls are wells with only cells and extra medium instead of a stimulus. They help ensure that cells do not respond without stimuli.

Question 30

Explain the concept of positive controls in the context of this experiment.

Answer 30

Positive controls are wells with cells and mitogen that activate macrophages to produce ROS. They are used to confirm cell responsiveness.

Question 31

How many times are the stimuli added to the wells, and why?

Answer 31

The stimuli are added 4 times to reduce measurement errors and assess data robustness. The average of the 4 rows is calculated to correct errors.

Question 32

Do you expect a difference in the location of formazan in cells of columns 3, 4, and 5? Why or why not?

Answer 32

No, formazan is expected to be located within the cells in all three columns. Column 3 is a known stimulant, but the difference between 4 and 5 is uncertain.

Question 33

Why are the four repetitions not identical in this experiment?

Answer 33

The repetitions vary due to factors like air bubbles, student errors, and cells settling at the bottom of the plate, which can influence measurements.

Question 34

Why is there blue coloration in A1 (first column first sample) in this experiment?

Answer 34

Blue coloration in A1 could indicate an error during liquid addition, such as accidentally adding macrophage suspension.

Question 35

Explain the significance of the baseline control in this experiment.

Answer 35

The baseline control, which includes cells but no stimulant, serves as a reference point to compare the effects of different stimuli on the cells.

Question 36

Describe the expected outcome in terms of blue coloration in the experiment.

Answer 36

More oxygen radicals produced will result in bluer cells. Positive control and columns with yeast and macrophages are expected to turn blue.