Cell Navigator® Fluorimetric Lipid Droplet Assay Kit *Green Fluorescence*

Protocol summary

1. Prepare cells with test compounds
2. Add Nile Green™ working solution
3. Incubate at room temperature or 37°C for 10 to 30 min
4. Read fluorescence intensity with fluorescence microscope using FITC filter

Important notes
Following is our recommended protocol for live cells. This protocol only provides a guideline, and should be modified according to your specific needs. Since Nile Green™ has minimal fluorescence in aqueous media, aspiration of the growth medium and removal of Nile Green™ staining solution after staining is optional. Stained cells can be fixed with 3 - 4% formaldehyde. In addition, prefixed cells (3 - 4% formaldehyde fixation) can be stained with Nile Green™ staining solution.

Prepare Nile Green™ working solution by diluting 5 µL of 200X Nile Green™ (Component A) to 1 mL of Staining Buffer (Component B). Note: 50 µL of Nile Green™ (Component A) is enough for one 96-well plate. Protect from light. The optimal concentration of the Nile Green™ varies depending on specific applications. The staining conditions may be modified according to a particular cell type and the permeability of the cells or tissues to the probe.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

For adherent cells:

1. Grow cells either in a 96-well black wall/clear bottom plate (100 µL/well/96-well) or on cover-slips inside a petri dish filled with the appropriate culture medium.
   
   
2. Gently aspirate the culture medium and add equal volume (such as 100 µL/well/96-well plate) of the Nile Green™ staining solution.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 10 - 30 minutes.
   
   
4. Remove Nile Green™ working solution (Optional).
   
   
5. Read Fluorescence at 485/520 nm with a microplate reader or observe the cells using a fluorescence microscope with a FITC filter set.
   
   

For suspension cells:

1. Centrifuge the cells at 1000 rpm for 5 minutes to get 1 - 5 × 10⁵ cells per tube.
   
   
2. Resuspend cells in 500 µL of Nile Green™ working solution.
   
   
3. Incubate at room temperature or 37°C for 10 to 30 min, protected from light.
   
   
4. Centrifuge to remove the Nile Green™ working solution, and resuspend cells in 500 µL of pre-warmed medium or buffer of your choice to get 1 - 5 × 10⁵ cells per tube (Optional).
   
   
5. Monitor the fluorescence increase using fluorescence microscope with a FITC filter set.