• Mesenchymal stem cells;
  • Apoptosis;
  • Flow cytometry;
  • Cell culture


  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References

Hui and colleagues have raised the concern that the detection of cell surface Annexin V staining on mesenchymal stem cells (MSC) following treatment with the enhancer of Zeste homology 2 (EZH2) inhibitor, DZNep (Sequoia Research, Pangbourne, United States, may have been compromised due to the use of Trypsin/EDTA to detach the adherent cells in our recent publication “EZH2 and KDM6A Act as an Epigenetic Switch to Regulate MSC lineage specification” [1]. Specifically, it is proposed that EDTA could affect the structure of the plasma membrane and chelate Ca2+ preventing the binding of Annexin V protein to the phosphatidylserine [2-4]. However, different manufacturers specify the use of Trypsin/EDTA, and not EDTA-free trypsin for detaching adherent cells prior to staining for Annexin V, where Trypsin/EDTA has been used by various studies for a wide range of adherent cell types [5-12].

While the Letter to the Editor by Hui and colleagues, published in this article, use the example that DZNep has been previously shown to induced apoptosis in human colon cancer stem cells [13], our study [1] found that DZNep had no effect on the apoptosis status of human bone marrow-derived MSC, in accord with other studies reporting that DZNeP is not harmful for nontransformed cells at tumor-inhibiting doses [14]. The discrepancy in DZNep sensitivity between normal stem cells and cancer stem cells may be due to aberrant levels of EZH2 in cancer cells required for survival [15, 16].

In order to determine whether EDTA influences Annexin V binding on MSC, we performed the Annexin V protocol with either Trypsin/EDTA (Sigma Aldrich, NSW, Australia, as described in our published study [1], or with EDTA-free Collagenase I (2 mg/ml Worthington Biochemical Corporation, NJ, United States of America, Two different human MSC cultures were treated with 5 µM of the potent apoptotic drug, doxorubicin (Sapphire Biosciences, NSW, Australia, for 24 hours prior to Annexin V staining. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then washed with Hanks Balanced Salt Solution (Sigma Aldrich, NSW, Australia, supplemented with 5% fetal calf serum (FCS; SAFC Biosciences, Sydney, NSW, Australia, The cells were further washed twice with cold phosphate buffered saline prior to cell staining for Annexin V as specified in the manufacturer's protocol (Annexin V conjugates Alexa Fluro 488, for apoptosis detection, Life Technologies, VIC, Australia, Flow cytometry was used to determine the levels of Annexin V staining under each condition. Cells treated with doxorubicin without Annexin V staining were used for negative gating. Flow cytometric analysis showed that there was no significant difference in the mean fluorescence levels between Trypsin/EDTA and collagenase I-treated MSC (Fig. 1). These results support recent findings by another group that reported no difference in Annexin V staining of apoptotic cells, following Trypsin/EDTA and Trypsin-free EDTA treatment, using two different adherent cell lines [6]. Therefore, we believe the use of Trypsin/EDTA for cell detachment has little influence on the measurement of early cell apoptosis by Annexin V staining on human MSC as we have previously reported [10].


Figure 1. Comparable expression of Annexin V on doxorubicin treated MSC detached by Trypsin/EDTA or Collagenase I. Two different human bone marrow-derived MSC cultures were treated with 5 µM doxorubicin for 24 hours. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then stained for Annexin V. Assessment of Annexin V levels was performed by flow cytometric analysis. Data represent mean fluorescence intensity from three replicate experiments. Abbreviation: MSC, mesenchymal stem cells.

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Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References

The authors indicate no potential conflicts of interest.

Author contributions

  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References

S.H.: experimental design, experimental procedures, data analysis, manuscript preparation; D.C.: experimental design, experimental procedures, data analysis, manuscript preparation; S.G.: project development, experimental design, data analysis, manuscript preparation, financial support.


  1. Top of page
  2. Rebuttal
  3. Disclosure of Potential Conflicts of Interest
  4. Author contributions
  5. References
  • 1
    Hemming S, Cakouros D, Isenmann S et al. EZH2 and KDM6A act as an epigenetic switch to regulate mesenchymal stem cell lineage specification. Stem Cells 2014;32:802815.
  • 2
    Hammill AK, Uhr JW, Scheuermann RH. Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic death. Exp Cell Res 1999;251:1621.
  • 3
    Saraste A. Morphologic criteria and detection of apoptosis. Herz 1999;24:189195.
  • 4
    Meers P, Daleke D, Hong K et al. Interactions of annexins with membrane phospholipids. Biochemistry 1991;30:29032908.
  • 5
    Borahay MA, Lu F, Ozpolat B et al. Mullerian inhibiting substance suppresses proliferation and induces apoptosis and autophagy in endometriosis cells in vitro. ISRN Obstet Gynecol 2013;2013:361489.
  • 6
    Xu XY, Nie XC, Ma HY et al. Flow cytometry method analysis of apoptosis: No significant difference between EDTA and EDTA-free trypsin treatment procedure. Technol Cancer Res Treat 2014;3:3.
  • 7
    Im W, Chung JY, Bhan J et al. Sun ginseng protects endothelial progenitor cells from senescence associated apoptosis. J Ginseng Res 2012;36:7885.
  • 8
    Al-Jamal KT, Kostarelos K. Assessment of cellular uptake and cytotoxicity of carbon nanotubes using flow cytometry. Methods Mol Biol 2010;625:123134.
  • 9
    Chang C, Liu SP, Fang CH et al. Effects of matrine on the proliferation of HT29 human colon cancer cells and its antitumor mechanism. Oncol Lett 2013;6:699704.
  • 10
    Kortesidis A, Zannettino A, Isenmann S et al. Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood 2005;105:37933801.
  • 11
    Fens MH, van Wijk R, Andringa G et al. A role for activated endothelial cells in red blood cell clearance: Implications for vasopathology. Haematologica 2012;97:500508.
  • 12
    Chavala SH, Kim Y, Tudisco L et al. Retinal angiogenesis suppression through small molecule activation of p53. J Clin Invest 2013;123:41704181.
  • 13
    Benoit YD, Witherspoon MS, Laursen KB et al. Pharmacological inhibition of polycomb repressive complex-2 activity induces apoptosis in human colon cancer stem cells. Exp Cell Res 2013;319:14631470.
  • 14
    Crea F, Fornaro L, Bocci G et al. EZH2 inhibition: Targeting the crossroad of tumor invasion and angiogenesis. Cancer Metastasis Rev 2012;31:753761.
  • 15
    Holland D, Hoppe-Seyler K, Schuller B et al. Activation of the enhancer of zeste homologue 2 gene by the human papillomavirus E7 oncoprotein. Cancer Res 2008;68:99649972.
  • 16
    Fluge O, Gravdal K, Carlsen E et al. Expression of EZH2 and Ki-67 in colorectal cancer and associations with treatment response and prognosis. Br J Cancer 2009;101:12821289.