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Fluorescence

Stem Cell CDy1 Dye

live stain for imaging and sorting pluripotent stem cells

Active Motif's Stem Cell CDy1 Dye provides a fast and simple method to selectively stain live pluripotent stem cells for identification and sorting using fluorescence microscopy and FACS. Because CDy1 is a live cell dye, it eliminates the need to sacrifice precious stem cell samples for testing, providing a significant advantage over other methods such as immunostaining that require cell fixation. In comparison to other live cell staining techniques, CDy1 is highly specific for pluripotent stem cells and does not produce background signal in other cells, such as feeder or differentiated cells, like live alkaline phosphatase staining methods. Staining is reversible and does not impact cell growth, morphology, or differentiation, making it ideal for stem cell enrichment and sorting, or for performing additional experiments or counterstaining procedures.

Unlike alkaline phosphatase live staining, CDy1 is specific for pluripotent stem cells.
Unlike alkaline phosphatase live staining, CDy1 is specific for pluripotent stem cells.

(Click image to enlarge)

Scientists at the Agency for Science, Technology and Research (A*STAR) and the National University of Singapore first discovered the dye as a selective stain for pluripotent mouse and human embryonic stem cells (mESCs, hESCs) enabling the identification stem cells from feeder cells or mixed cultures. In these studies, the CDy1 dye was also utilized to determine reprogramming of induced pluripotent stem cells (iPSC) following retroviral infection of the Oct4, Sox2, Klf4 and c-Myc transcription factors. Their studies were published in Nature Protocols in 20111. Subsequent studies validating the use of the Stem Cell CDy1 dye for identification and selection of other stem cell types have also been published. In a recent publication from the laboratory of Geoffrey W. Osborne at the University of Queensland, the Stem Cell CDy1 dye was used to enrich for neural stem cells from mouse brain2.  Also, studies from the laboratory of Robert Hawley demonstrate the use of the Stem Cell CDy1 dye to identify a subpopulation of drug-resistant Multiple Myeloma (MM) cells believed to be cancer stem cells2. To see detailed information on published data utilizing the CDy1 Dye, please click on the References tab below.

  1. Kang, N.Y. et al. (2011) Nat. Protoc. 6, 1044-1052.
  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.
Flow cytometry dot-plot image of mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) stained with Stem Cell CDy1 Dye
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). Protocol information for flow cytometry analysis of CDy1 stained mESCs is referenced in Kang N.-Y. et al. (2011) Nat Protoc.

Fluorescent images of mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) stained with Stem Cell CDy1 Dye and Oct4 antibody
Figure 2: 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 Oct4 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 Oct4 (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.

 
Name Format Cat No. Price  
Stem Cell CDy1 Dye 50 µl 14001 $300 Buy Now

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.

Fluorescent images of mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) stained with Stem Cell CDy1 Dye and Oct4 antibody
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.
Flow cytometry dot-plot image of mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) stained with Stem Cell CDy1 Dye
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). Protocol information for flow cytometry analysis of CDy1 stained mESCs is referenced in Kang N.-Y. et al. (2011) Nat Protoc.

Fluorescent images of hESCs,mESCs and iPSCs stained with Stem Cell CDy1 Dye
Figure 2: Stem Cell CDy1 Dye Selectively Stains Pluripotent Stem Cells.

Human embryonic stem cells (hESC), mouse embryonic stem cells (mESC) and induced pluripotent stem cells (iPSC) were grown on feeder cells and stained without fixation with either DMSO as a negative control or Stem Cell CDy1 dye. The cells were then visualized by fluorescent microscopy. The images show selective staining of all types of embryonic stem cells (hESCs, mESCs and iPSCs) within the mixed cell cultures by the CDy1 dye. Furthermore, punctate staining is observed within the formed stem cell colonies. This staining pattern results from the CDy1 dye selectively staining pluripotent cells, and not differentiated cells, within the colonies as some of the cells have already begun the process of early stage differentiation. The DMSO stain of hESCs is provided as a representative negative control image. These images were kindly provided courtesy of Rhoda Mondeh-Lowor at California State University, San Marcos.

Fluorescent images of human induced pluripotent stem cells (hiPSCs) and mouse embryonic fibroblasts (MEF) stained with Stem Cell CDy1 Dye
Figure 3: 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.

Fluorescent images of mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) stained with Stem Cell CDy1 Dye and Oct4 antibody
Figure 4: 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 Oct4 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 Oct4 (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.

CDy1 dye shows specificity for pluripotent stem cells compared to alkaline phosphatase staining
Figure 5: CDy1 dye is specific for pluripotent stem cells unlike alkaline phosphatase staining methods.

Human embryonic stem cells (hESC), mouse embryonic stem cells (mESC), and induced pluripotent stem cells (iPSC) were grown on feeder cells and stained without fixation with either Stem Cell CDy1 dye (CDy1, red) or alkaline phosphatase (ALP, green) using a competitor's live ALP staining kit. The images show that, unlike alkaline phosphatase staining methods, CDy1 is highly specific for pluripotent stem cells and does not produce background signal in other cells, such as feeder or differentiated cells. In contrast, alkaline phosphatase staining only differentially stains pluripotent cells at higher levels but still produces background staining in feeder cells and differentiated cells. These images were kindly provided courtesy of Rhoda Mondeh-Lowor at California State University, San Marcos.

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:

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.