Cell Tracking Red Dye Kit - Longer cell staining, DMSO-free

Cell Tracking Red Dye Kit ab269446 stains cell membranes in live cells, and is ideal for use in cell tracking and cell labeling. It uses SomaServe's PolyNaut^® dye-loading technology to enable long-term live cell staining, and is used without DMSO in order to minimise cell toxicity.

Longer cell staining than the leading competitor Cell Tracking Red dye, with less impact on cellular behavior

In cell tracking experiments with PolyNaut dye-loaded ab269446 (see image section), cells remain viable for up to 20 days, unlike with the leading competitor Cell Tracking Red dye where cell viability rapidly decreases after ~5 days.

- stain cells for cell tracking for up to 20 days
- incubate with dye for as little as 30 min to stain cells, or leave dye in cell culture media for 96 hours or more without effect on cell viability
- load dyes into cells without DMSO; which is associated with cell toxicity and is likely to disturb cellular behavior even when used at low levels
- 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 DMSO and lower dye concentration
- stain the entire cell structure (with weaker staining of nuclei, allowing nuclei to be identified by the absence of stain)
- easily restain cells by adding more dye solution

The dye is supplied in a ready-to-use format and is based on Rhodamine B (Ex max 554 nm, Em max 575 nm).

PolyNaut dye-loading enables long period cell tracking without toxicity

The Cell Tracking Red Dye 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 Cell Tracking Red Dye Kit ab269446

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 ab269446 1:20 with fresh cell culture medium; adding 1 part of ab269446 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, ab269446 may be added directly to the media (e.g. 5 μL of ab269446 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 554 nm, Em max 575 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 ab269446 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.