NBD Cholesterol Staining Dye Kit ab269448 can be used to study the mechanisms and biological factors that regulate cholesterol movement within cells. NBD Cholesterol is a fluorescently labelled cholesterol.

This kit uses SomaServe's PolyNaut™ dye-loading to load NBD cholesterol into live cells in cell cultures. Traditionally, NBD cholesterol is applied to cell cultures dissolved in ethanol, with a final concentration of 0.5%-2% ethanol in the cell culture, which is close to the levels of ethanol associated with cellular toxicity. PolyNaut dye-loading does not require ethanol.

NBD cholesterol with PolyNaut dye-loading

With the PolyNaut dye-loaded NBD Cholesterol:

- incubate with dye for as little as 30 min to stain cells, or leave dye in cell culture media for up to 24 hours without effect on cell viability
- load dyes into cells without ethanol; which is associated with cell toxicity
- reduce toxicity from the cell staining dye as polymersome dye-loading can be used with very low dye concentrations
- disturb cell behaviour less due to the lack of ethanol and lower dye concentration
- easily restain cells by adding more dye solution

The dye is supplied in a ready-to-use format. The NBD cholesterol used is 22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3β-Ol with CAS number 78949-95-8. NBD cholesterol stains cells a green color and has peak excitation at 469 nm and peak emission at 537 nm.

PolyNaut dye-loading mechanism

The NBD cholesterol in this kit is supplied encapsulated in SomaServe's PolyNaut™ particles. PolyNaut particles are non-toxic, pH-sensitive vesicles which are used to deliver cargos into the cell without the need for solvents and with no effect on metabolic activity.  

The dye-loaded particles are taken into the cell by endocytosis. Dye is then released from the particles in the early endosome. It then escapes into the cytosol enabling cell staining.

PolyNaut particles enable dyes to be used in forms which do not cross the cell membrane, minimizing transfer of cell tracking and other dyes between cells. Many publications have been made using dye-loaded PolyNaut particles for live cell staining; a deeper explanation of the technology is found in Massignani M et al (2010) PLoS One 5(5):e10459.

Live cell staining protocol for NBD Cholesterol Staining Dye Kit ab269448

1.) For adherent cells, culture cells on surface that is compatible with available imaging system (e.g. coverslip, chamber slide, clear-bottom optical multiwell plates, etc). For a 96-well plate, an initial recommendation is to use 10E4 - 10E5 cells per well. Please note, that the optimal cell density needs to be determined experimentally for each assay.

Suspension cells are prepared similarly as adherent cells but with centrifugation (200g, 5 minutes) before and after each washing step.

2.) To prepare the labelling solution, dilute ab269448 1:20 with fresh cell culture medium. Serum can be present in the medium, as it will not interfere with the labelling. The recommended volume to use with a 96-well plate is 100 µl per well. In this example, 5 µl are mixed with 95 µl of fresh cell culture medium. For live cell imaging, usage of phenol red-free cell culture medium is recommended.

Note: As alternative to preparing a labelling solution as described above, ab269448 may be added directly to the media (e.g. 5 µL in 100 µL cell culture media). In this case, please proceed with step 4.

3.) Add labelling solution to the well.

4.) Incubate at 37 °C, 5% CO₂, for 30 minutes to 24 h.

Note: Because the uptake of the polysomes and release of the dye takes time, the labelling generally increases within the first 2 h. The optimal incubation time depends on the cell type, i.e. tumour cells uptake is usually faster than primary cells. As a minimum, we recommend 30 min incubation with labelling solution. We do not recommend imaging longer than 24 h, because the fluorescence decreases rapidly after this time.

5.) Labelled cells can be observed by microscopy while they are in labelling solution. Alternatively, the labelling solution can be removed at any given time point by 3x washing with fresh culture media. If the labelling solution is removed, cells remain labelled for at least 1h, but generally not longer than 24 h.

6.) Optional: If DNA counterstaining with Hoechst 33342 (ab228551) is required, we recommend diluting ab228551 at 1:10000 in cell culture media and to incubate for 15 min. Wash cells two times with pre-warmed fresh cell culture medium.

7.) If required, cells labelled with ab269448can be fixed with 4% PFA (although this tends to shrink the cells and make it harder to achieve high quality images). Note that MeOH fixation is not compatible with this product.