Do you know what respiratory burst is and how to measure it? Find a complete protocol for Respiratory Burst Activity Measurement, using DHR-123, fluorescent probe.

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What is that?

Apart from the damage it can cause, oxidative stress can also play an important role in the functioning of organisms, especially in the immune response and inflammation processes. An example is the respiratory burst, the intended production of free radicals from cells belonging to the immune system, like neutrophils and monocytes, when getting in contact with pathogens. The reactive oxygen and nitrogen species yielded from this process, help in the digestion of the leftover particles of bacteria and fungi that have been phagocyted. Impairment in the functioning of the respiratory burst causes high infection rates or tissue damage and leads to clinical complications.

Interesting, so how can you measure it?

One of the most reliable methods is simply by using the DHR123 probe (available in BQCKit). This compound will become fluorescent when exposed to radical species, and the best part is that it can enter the cell! The fluorescence is typically measured by flow cytometry to increase the accuracy of the assay.

What other reagents will you require for this assay?

  • Assay Buffer: is used to suspend the cells into. It consists on RPMI 1640 base medium mixed with BSA and calcium chloride.
  • PMA: this chemical compound is able to stimulate the production of ROS by the NADPH oxidase and increase the fluorescence of the samples as a positive control.
  • Red Blood Cell Lysis Buffer: used to remove red blood cells and its interferents (e.g. haemoglobin) to isolate leukocytes in whole blood.

How to measure the respiratory burst activity?

Find a complete protocol for Resporatory Burst Activity Measurement, using DHR-123, fluorescent probe.

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Assay protocol

1 Add 10 µl of sample to a tube
2 Add 10 µl of DHR123 to the tube (available at Bioquochem)
3 15 min Incubate at 37ºC
4 Add 25 µl of PMA/PBS/other
5 45 min Incubate at 37ºC
6 Add 2 ml of Red blood cell lysis buffer
7 3-20 min Incubate at 37ºC
8 10 min Centrifuge at 500 xg at RT
9 Remove supernatant
10 Resuspend cell pellet in 0.5 ml assay buffer
11 Measure by flow cytometry with a fluorescence emission at 530 nm

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