BODIPY TR Ceramide Golgi Staining Kit ab269449 can be used to stain Golgi apparatus in live cells and study sphingolipid transport and metabolism mechanisms. The BODIPY TR Ceramide dye is supplied ready-to-use. BODIPY TR Ceramide is a red/orange color, with peak excitation at 589 nm and peak emission at 616 nm.

This kit uses SomaServe's PolyNaut™ dye-loading to load BODIPY TR Ceramide into live cells in cell cultures. 

With the PolyNaut dye-loaded BODIPY TR Ceramide:

- 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
- easily restain cells by adding more dye solution
 

PolyNaut dye-loading mechanism

The BODIPY TR Ceramide 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 BODIPY TR Ceramide Golgi Staining Kit ab269449

1) Culture adherent cells on a surface that is compatible with available imaging systems (e.g. coverslip, chamber slide, clear-bottom optical multiwell plates, etc. Suspension cells should be prepared similarly, with centrifugation (200g, 5 minutes) before and after each washing step.

Determine the optimal cell density / number for each assay experimentally; 10⁴ - 10⁵ cells per well is an initial recommendation if using a 96-well plate.

2) To prepare the labeling solution, dilute ab269449 1:20 with fresh cell culture medium; adding 1 part of ab269449 to 19 parts of medium.

Serum in the medium will not interfere with the labeling. We recommend 100 µl of labeling solution per well if using a 96-well plate. For live cell imaging, use phenol red-free cell culture medium.

3) Add labelling solution to the well.

Instead of preparing a labelling solution, ab269449 may be added directly to the media (e.g. 5 µL of ab269449 added to 100 µL of cell culture media).

4) Incubate at 37 °C, 5% CO₂, for 30 minutes to 96 h.

Labelling generally increases over the first 10 h as uptake of the polysomes and release of the dye takes time. The optimal incubation time depends on cell type, eg tumor cells usually uptake faster than primary cells. As a minimum, we recommend 30 min incubation with labelling solution. If cells are not sufficiently labelled after this time, we recommend increasing the labelling time to 2h, 10 h, etc., up to 96 h.

5) Labelled cells can be observed by microscopy (Ex max 589 nm, Em max 616 nm) 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. After removing the labelling solution, depending on cell type, cells will remain labelled for up to 6 days or more.

Optional: If DNA counterstaining with Hoechst 33342 (ab228551) is required, we recommend diluting ab228551 at 1:10,000 in cell culture media and incubating for 15 min. Wash cells two times with pre-warmed fresh cell culture medium.

6) If required, cells labelled with ab269449 can 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.