Stem Cell CDy1 Dye

Stem Cell CDy1 Dye Advantages

• Works on live cells, no need for fixation or cell sacrifice
• Simple, fast protocol for identification of stem cells from feeder cells or mixed cultures
• CDy1 Dye does not affect the stem cells ability to differentiate into lineage-specific cell lines
• Immunostaining and counterstaining procedures can be performed on the same cells following CDy1 staining
• Can be used to determine iPSC reprogramming earlier than genetic reporter systems
• Easy addition of dye to cell culture medium enables higher throughput screening
• Works with common fluorescent filter sets (Ex. 544 nm, Em. 577 nm)

CDy1 Background

The CDy1 dye was developed as part of a collaboration including the Agency for Science, Technology and Research (A*STAR) in Singapore. Researchers screened the Diversity-Oriented Fluorescence Library (DOFL) to identify novel small-molecule imaging probes specific for pluripotent stem cells and discovered the fluorescent rosamine compound, CDy1, could selectively stain live ESCs and iPSCs¹. The stain could be used to identify and isolate ESCs from a mixture containing MEF feeder cells. Additional characterization of CDy1 revealed that CDy1 can be used to determine iPSC reprogramming at 10 days post retroviral infection of the Oct-4, Sox2, KLF4 and c-Myc transcription factors, while the GFP reporter system did not show expression of GFP until 17 days post retroviral infection, indicating that Stem Cell CDy1 Dye can be used to determine iPSC reprogramming at earlier time points than genetic reporter systems¹.

Conventional methods used to identify stem cells include characterization by colony morphology, screening for expression of alkaline phosphatase and immunostaining for stem cell markers. Stem Cell CDy1's ability to stain living cells provides a significant benefit over methods such as immunostaining and alkaline phosphatase detection which require cell fixation, thereby rendering the cells unusable for future experiments. Another commonly used staining method, the Aldefluor method, uses BODIPY-aminoacetaldehyde, which is a substrate of aldehyde dehydrogenase, to stain hematopoietic stem cells and some cancer stem cells. A comparison of the CDy1 dye and the Aldefluor stain revealed that Aldefluor does not stain ESCs¹. The CDy1 dye has also been shown by subsequent published studies to be selective for other stem cell types, including neural stem cells, as shown by work from the laboratory of Dr. Geoffrey W. Osborne at the University of Queensland², and cancer stem cells, as demonstrated by research from the laboratory of Dr. Robert Hawley at George Washington University³.

The drawbacks and limitations presented by conventional methods makes Active Motif's Stem Cell CDy1 Dye an ideal solution for the identification and isolation of stem cells. With CDy1's ability to stain living cells, there is no longer a need to sacrifice precious stem cell samples. Cells that have been stained will have the same growth rate, morphology and differentiation capability as unstained stem cells. Simply add Stem Cell CDy1 Dye to the culture medium of living cells. Following a 1 hour incubation and 3 hour destaining, cells are washed and prepared for imaging⁴. The spectral properties of the CDy1 dye enables detection of stained cells using fluorescence microscopy with TRITC or Cy3 filter sets, while a 488 nm laser and a PE-Texas Red filter can be used for analysis with flow cytometry⁴.

Figure 1: CDy1 Staining Coincides with Expression of Stem Cell Markers.

Mouse embryonic stem cells (mESCs) grown on a mouse embryonic fibroblast (MEF) feeder layer were stained without fixation with Stem Cell CDy1 dye. CDy1 staining was visualized by fluorescent microscopy. The cells were then fixed in 4% paraformaldehyde, stained with Oct-4 antibody and visualized with FITC-conjugated secondary antibody. The images show that mESC aggregates, but not MEF feeder cells, stain positive for both CDy1 (red) and Oct-4 (green). Scale bar, 100 μm. The images were kindly provided courtesy of Dr. Y-T Chang at the National University of Singapore, Republic of Singapore.

References

1. Im, C.N. et al. (2010) Angew. Chem. Int. Ed. Engl., 49:  7497-7500.
2. Vukovic, J. et al. (2013) Stem Cells Dev. doi:10.1089/scd.2012.0660.
3. Hawley, T.S. et al. (2013) Am J Hematol. 88, 265-272.
4. Kang, N.Y. et al. (2011) Nat. Protoc., 6:  1044-1052.

Figure 1: FACs analysis of mESCs and MEFs stained by Stem Cell CDy1 Dye.

Mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) were stained with the Stem Cell CDy1 Dye and then analyzed by flow cytometry (FACS) using side scatter (SSC) and a PE-Texas-Red channel. CDy1 enables clear differentiation and isolation of the pluripotent mESCs (red) vs. the MEFs (blue).

Figure 2: Stem Cell CDy1 Dye Selectively Stains iPSCs in Live Mixed Cell Populations.

Human induced pluripotent stem cells (hiPSCs) derived from umbilical cord blood were grown on a mouse embryonic fibroblast (MEF) feeder layer and stained without fixation with Stem Cell CDy1 dye. The cells were then visualized using phase contrast and fluorescent (TRITC) filters. The images show selective staining of hiPSC aggregates within the mixed cell culture by the CDy1 dye. These images were kindly provided courtesy of Dr. H. Zaehres at the Max Planck Institute for Molecular Biomedicine in Germany.

Figure 3: CDy1 Staining Coincides with Expression of Stem Cell Markers.

Mouse embryonic stem cells (mESCs) grown on a mouse embryonic fibroblast (MEF) feeder layer were stained without fixation with Stem Cell CDy1 dye. CDy1 staining was visualized by fluorescent microscopy. The cells were then fixed in 4% paraformaldehyde, stained with Oct-4 antibody and visualized with FITC-conjugated secondary antibody. The images show that mESC aggregates, but not MEF feeder cells, stain positive for both CDy1 (red) and Oct-4 (green). Scale bar, 100 μm. The images were kindly provided courtesy of Dr. Y-T Chang at the National University of Singapore, Republic of Singapore.

Stem Cell CDy1 Dye

To learn more about Active Motif's Stem Cell CDy1 Dye, including protocols details for staining and imaging of different stem cell types as well as application data using fluorescent imaging and FACS analysis, please refer to the references listed below:

“Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1.” by Kang, N.Y. et al. (2011) Nat. Protoc., 6:  1044-1052.

 

“A fluorescent rosamine compound selectively stains pluripotent stem cells.” by Im, C.N. et al. (2010) Angew. Chem. Int. Ed. Engl., 49:  7497-7500.

 

“A fluorescent screening platform for the rapid evaluation of chemicals in cellular reprogramming.” by Vendrell, M. et al. (2012) Stem Cell Res., 9:  185-191.

 

“Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1.” by Hawley, T.S. et al. (2013) Am J Hematol., 88:  265-272.

 

“A novel fluorescent reporter CDy1 enriches for neural stem cells derived from the murine brain.” by Vukovic, J. et al. (2013) Stem Cells Dev., doi:10.1089/scd.2012.0660.

 

Contents & Storage

Stem Cell CDy1 Dye is supplied as a 50 µl stock solution in DMSO. We recommend making 10 µl aliquots upon first use of the dye and storing at -20°C.