Product nameCell 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.
Storage instructionsShipped at 4°C. Store at +4°C.
Storage bufferpH: 7.40
Constituents: 0.00005% Rhodamine B octadecyl ester perchlorate, 0.2% Sodium hydroxide, 89.9% PBS, 0.01% Polymer PMPC-PDPA
Concentration information loading...
Comparison of ab269446 Cell Tracking Red Dye with leading competitor Cell Tracking Red dye
Human dermal fibroblasts (HDF) were seeded in a glass-bottom 24-well black plate (Ibidi) at 10,000 cells per well and incubated overnight in a humidified atmosphere 5% CO2 at 37 °C. ab269446 was prepared by dilution in fibroblast growth medium (PromeCell) at 1/20, 1/40 and 1/60 ratio, respectively. HDF cells were then treated with the previously prepared solutions of ab269446 and a leading competitor's dye at 5 µM dissolved in DMSO. After 1, 2, 4, 6, 8, 10, 15 and 20 days of incubation in a humidified atmosphere 5% CO2 at 37 °C, HDF cells were washed 3-times with phosphate-buffered saline (PBS) and then 500 µL/well of cell culture medium was added. Fluorescence in the treated HDF cells was accessed by live confocal fluorescent scanning microscopy (CLSM) using a Leica TCS SP8 microscope in an atmosphere at 37 °C and CO2.
Immediately after live cell imaging, HDF cells were incubated with 0.5 mg/mL of 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) for 2 hours in a humidified atmosphere 5% CO2 at 37°C. Intracellular metabolic activity reduces MTT solution to a purple formazan crystal salts, which were then solubilised by adding 200 µL/well of DMSO. After 10 minutes incubation at room-temperature protected from light, the optical density at 490 nm was then measured using a UV/Vis spectrophotometer microplate reader (BioTek). The metabolic activity of cells and hence the cell viability was determined by normalising the absorbance measurements of each treatment well with the control well (i.e. non-treated HDF cells).
Left Image: Confocal analysis. The cells were treated for 24 hours with either ab269446 1/20 dilution or leading competitor's dye and then washed with PBS and left in complete medium up to 20 days.
Right Image: Cytotoxicity analysis. The cells have been treated for 24 hours with either ab269446 1/20 dilution or leading competitor's dye (5µM) and then washed with PBS and left in complete medium up to 20 days.
ab269446 allow the imaging of cells with high contrast details and fluorescence up to 20 days. This is the results of high delivery capability of the technology coupled with receptor mediated uptake.
Cells cultured for 10 days with only one single exposure to ab269446 for 24 hours. The cells are than passaged resulting with the fluorescence successfully retained.
Primary human dermal fibroblast cells stained with Cell Tracking Red Dye ab269446. Cells were imaged at 1, 7 and 14 days for the same exposure, after a single dose of dye.
Cell Tracking Red Dye ab269446 was applied to primary mouse splenocytes for 48 hrs.
The dye was added directly into the media at a 1:5 dilution. Images were taken 2 h, 8 h, 14 h, 24 h and 48 h after application of the dye.
Cells were cultured in splenocyte culture medium (RPMI, 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1x non-essential amino acids, 0.055 mM beta-mercaptoethanol, 1x antibiotic-antimycotic solution). Image data was acquired with the Perkin Elmer Operetta HCA (37°C, 5% CO2). Confocal images are shown with identical image acquisition settings at each time point.
Confocal laser scanning microscopy 3D reconstruction of a primary human dermal fibroblast after treatment with Cell Tracking Red Dye ab269446, a lysosomal staining dye, and a green fluorescent DNA staining dye.
Rhodamine B loaded polymersomes used to examine the migration of primary human dermal fibroblast cells between a seeding area and a pre-cast fibrin clot gel.
(a) Imaged after 7 days at the initial cell seeding area. (b) Imaged after 7 days at the interface between the initial cell seeding area and the pre-cast gel. (c) Imaged after 14 days at the initial cell seeding area. (d) Imaged after 14 days at the interface between the initial cell seeding area and the pre-cast gel. Figure bar = 0.1 mm.
Staining with Rhodamine B loaded polymersomes allows visualization of fine structural details of live cells.
Image shows 3D reconstruction from confocal laser scanning optical stacks of primary rat motor neuron cells from Massignani M et al 2010.
Cell Tracking Red Dye ab269466 has been used to stain many cell types including:
- primary rabbit limbal epithelial cells
- primary rat cortical neurons and motor neurons
- primary human dermal fibroblasts, epidermal keratinocytes, endothelial cells, monocytes, macrophages, MSc stem cells
- KB and SCC4 H&N cancer cells and preosteocytes
- CHO cells
- rat swanoma cells
Cell Tracking Red Dye ab269446 was applied to A375 cell cultures for 24 hrs. Cell media was changed at 24 hrs, removing the dye.
Image is at 96 hrs (4 days) after application of the dye.
Cells were cultured in phenol-red free DMEM with 10% FBS. Image data was acquired with the Perkin Elmer Operetta HCA (37°C, 5% CO2).
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab269446 has been referenced in 2 publications.
- Kung KS et al. The development of anisotropic behaviours of 3T3 fibroblasts on microgrooved patterns. Eur Phys J E Soft Matter 34:23 (2011). PubMed: 21380646
- Massignani M et al. Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes. PLoS One 5:e10459 (2010). PubMed: 20454666