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Practical report immunology
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NBT assay in short:1) Macrophages (RAW cells) added in a 96-wells plate (105 cells per well) are cultured overnight to
adhere to the bottom of the well.
2) The culture medium is replaced by NBT solution (formazan salt). NBT diffuses into the cell.
3) Stimuli (e.g. pathogens) are added, and RAW cells are activated to produce oxygen radicals.
4) NBT is turned into the blue coloured formazan (solid) by oxygen radicals.
5) The cell membrane is permeabilized and solubilized by methanol and KOH.
6) The released solid formazan is dissolved by DMSO.
7) The color reaction can be measured in a spectrophotometer
Measuring the capacity of
macrophages to produce oxygen
radicals by applying an NBT
assay
Macrophages are mononuclear leukocytes of the innate immune system present in tissues. Before
the cell leaves the blood and becomes a macrophage, it is called a monocyte.
First, macrophages will recruit other cells to the site of infection. Then, the microbe may bind to the
pattern recognition receptor (PRR) of the macrophage, enabling phagocytosis. After degrading the
pathogen into peptides via free radicals, these smaller peptides are presented on the cell surface via
MHC class II molecules to Th cells (CD4+), which are cells of the adaptive immune system. These
antigen presenting cells (APC) are mostly present in lymphoid organs.
After recognizing a pathogen via TLR, the macrophage increases its O2
uptake. O2 is converted intracellular into O2- (superoxide anion) by using
NADP or NADH. The super oxide dismutase enzyme converts O2- into
H2O2. Together O2- and H2O2 exert strong microbial activity. The capacity
of macrophages to produce O2- will be measured by using NBT (Nitroblue
tetrazolium). O2- converts NBT into blue formazan; a spectrophotometer
can measure this response.
The aim is to determine the conversion of O2 into O2- by measuring blue formazan with a
spectrophotometer.
1. Cells were plated in a 96-wells plate and cultured overnight. Colum 1 only has medium
(blanc), column 2 till 5 are filled with macrophage cell suspension (100 µl)
2. The culture medium of the macrophages was washed away, 100 µl fresh RPMI medium
should be added quickly to avoid a stress reaction of the cells. Washing ensures comparable
conditions for each well.
3. The medium was again removed and all wells were filled with 160 µl NBT
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