Practical NBT assay AI generated Flashcards
Describe the NBT assay in the context of studying macrophages.
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.
What is the role of macrophages in the immune system?
Macrophages belong to the innate immune system and have a general clearance function, often referred to as ‘scavenger macrophages’.
How is the color reaction in the NBT assay measured?
The color reaction is measured in a spectrophotometer after the released solid formazan is dissolved by DMSO.
Define the primary immune response initiation involving macrophages.
It includes phagocytosis of antigens, degradation of pathogens to antigens, and presentation of antigenic determinants to CD4+ Th cells.
What are some functions of macrophages in the body?
Macrophages act as garbage collectors, removing around 10^11 red blood cells daily in humans and playing a crucial role in immune responses.
Describe the role of monocyte-derived macrophages in immune responses.
Monocyte-derived macrophages play a crucial role in the initiation phase of immune responses.
What is the function of Fc-receptors on macrophages’ surface?
Fc-receptors on macrophages’ surface allow them to efficiently clear immune complexes (antigen-antibody complexes).
How do macrophages present antigenic determinants to T helper cells in lymphoid organs?
Macrophages present antigenic determinants via MHC class II molecules and interact with T helper cells in lymphoid organs like the spleen.
Define phagosome and phagolysosome in the context of macrophages.
Phagosome is the membrane enveloping bacteria, while phagolysosome is where bacteria undergo degradation within macrophages.
What are some characteristics of the endosomal compartment of macrophages?
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.
How do phagocytes generate an ‘oxidative burst’ upon stimulation?
Phagocytes generate an ‘oxidative burst’ by increasing O2 uptake, converting O2 into superoxide anion, and further converting it to H2O2, which exhibit microbicidal activity.
Describe the process of culturing macrophages in an experiment.
In the experiment, macrophages adhere to the culture surface, are cultured overnight, and then washed for further analysis.
Describe the importance washing cells in a cell culture assay.
Washing cells helps standardize culture conditions, ensuring cells start under comparable conditions and preventing unwanted reactions.
How is the conversion of O2 to the superoxide anion measured in a cell culture assay?
It can be measured using Nitro-Blue-Tetrazolium, which is converted by the superoxide anion to a blue solid formazan, measurable with a spectrophotometer.
Define positive and negative controls in a cell culture assay.
Positive control involves using a known stimulus to elicit a specific reaction, while negative control involves having no cells present.
What is the purpose of examining the effect of different yeast concentrations in a cell culture assay?
To observe how macrophages phagocytose yeast and understand the impact of yeast concentration on the process.
Describe the potential consequences of wells being left without medium for too long in a cell culture assay.
Leaving wells without medium for an extended period can cause stress to the cells.
How does Nitroblue tetrazolium (NBT) contribute to a cell culture assay?
NBT diffuses into the cell and is converted by the superoxide anion to a blue solid formazan, aiding in measurement.
What happens if all reagents are combined without washing cells in a cell culture assay?
The solution turns blue without the presence of macrophages at the end, affecting the accuracy of the assay.
Describe the behavior of macrophages in a cell culture assay regarding the bottom of the plate.
Macrophages tend to stick to the bottom of the plate and form a cell layer, requiring careful handling to maintain the cell layer integrity.
Describe the process of removing excess NBT from the culture in the given content.
Excess NBT is removed by adding methanol to fix the cells and stop the reaction, allowing the amount of formazan produced to be determined.
What is the purpose of adding KOH in the content’s process?
KOH is added to permeate the cell membrane and allow DMSO to dissolve the blue formazan particles for coloration measurement.
Define the role of a spectrophotometer in the context of the content.
A spectrophotometer, also known as an ‘ELISA-reader,’ measures color intensity using filters corresponding to specific wavelengths.
How are the absorption results interpreted in the given content?
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.
