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Cat. No.: NRSK-1k (for 1000T)

Cat. No.: NRSK-5k (for 5000T)

Description

Lipid Droplets Red Fluorescence Assay Kit with Nile Red, also known as the Nile Red Staining Kit or Red Neutral Lipid Stain, is a reagent kit used for the detection of lipid droplets within cells based on the fluorescent probe Nile Red. This assay kit is suitable for fluorescence microscopy, flow cytometry, fluorescence plate readers, and other fluorescence detection systems.

Lipid droplets are composed of a neutral lipid hydrophobic core, consisting of a monolayer of phospholipids, triglycerides, and cholesteryl esters (CE), and are widely found in animal, bacterial, yeast, plant, and insect cells. Lipid droplets can move along the cell's cytoskeleton, interact with other organelles, and play essential roles in lipid metabolism and storage, membrane transport, protein degradation, and signal transduction processes. Various metabolic disorders such as obesity, fatty liver disease, cardiovascular diseases, diabetes, neutral lipid storage diseases, and Niemann-Pick C disease may be associated with abnormal lipid storage.

The fluorescent probe used in this assay kit is Nile Red, also known as Nile blue oxazone, with CAS number 7385-67-3 and a molecular weight of 318.37. Its maximum excitation wavelength in phospholipids is approximately 552nm, and its maximum emission wavelength is around 636nm, resulting in red fluorescence. It can also be used with an excitation wavelength set between 450-500nm and an emission wavelength greater than 528nm to produce green fluorescence. However, red fluorescence is generally much brighter than green fluorescence. Since Nile Red can also stain cell membranes when emitting red fluorescence, and it specifically stains lipid droplets when emitting green fluorescence, you can set the parameters to Ex/Em=485/535nm for specific lipid droplet detection if needed.

Nile Red exhibits almost no fluorescence in water and other polar solvents. However, once it binds to neutral lipids like triglycerides, it emits bright red fluorescence (due to its wide emission spectrum, parameters can also be adjusted to observe green fluorescence).

For samples in a 96-well plate, based on using 100μl of staining solution per well, the small and medium-sized packaging of this assay kit can be used for 1000 and 5000 detections, respectively. If used for flow cytometry with a detection system volume of 0.5ml per sample, the small-sized packaging of this assay kit can be used for 200 detections, and the medium-sized packaging for 1000 detections. For adherent cell samples in a 6-well plate, considering the use of 1ml of staining solution per well, the small and medium-sized packaging of this assay kit can be used for 100 and 500 detections, respectively.

Components

• Nile Red (1000X)
• Hoechst 33342 (1000X)
• Assay Buffer

Storage

Store at -20°C in the dark, with a minimum shelf life of one year. Nile Red (1000X) and Hoechst 33342 (1000X) should be protected from light.

Precaution

• Due to the broad emission spectrum of Nile Red, which includes both green and red fluorescence, it is advisable to minimize other fluorescent staining and detection as much as possible. If dual staining is necessary, because Nile Red rapidly quenches under laser illumination, it is recommended to first detect Nile Red and then perform other fluorescence detection after quenching.
• For the first-time usage, it is recommended to aliquot appropriately to avoid repeated freeze-thaw cycles.
• The working concentration of Nile Red, cell quantity, incubation temperature, and time can be optimized according to the specific cell type being used. For example, the concentration of Nile Red can be adjusted within the range of 0.2X to 2X.
• Nile Red is prone to quenching under laser illumination; therefore, it is necessary to reduce the dye concentration as much as possible while maintaining fluorescence brightness. This can be achieved by reducing the excitation light intensity of the fluorescence microscope and shortening the exposure time during imaging.
• All fluorescent dyes are susceptible to quenching, so please take precautions to protect them from light to minimize fluorescence quenching.
• This product is intended for use in scientific research by trained professionals and should not be used for clinical diagnosis or treatment, food, or pharmaceutical purposes. It should not be stored in a regular household setting.
• For your safety and health, please wear laboratory attire and disposable gloves while handling.

Only for research and not intended for treatment of humans or animals

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