Es cell characterization kit

Manufactured by Merck Group

Sourced in United States

The ES Cell Characterization Kit is a laboratory product designed to assist in the identification and analysis of embryonic stem (ES) cells. The kit provides a set of reagents and protocols for evaluating key characteristics of ES cells, such as the expression of specific marker proteins. This enables researchers to assess the pluripotency and differentiation potential of ES cell samples. The kit is intended to facilitate the characterization of ES cells within a research or laboratory setting.

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6 protocols using es cell characterization kit

Immunohistochemical Characterization of iPSCs

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For iPSC immunohistochemistry, Oct4, Sox2, Nanog, SSEA4, TRA-1-60 and TRA-1-81 (dilution 1: 50 to 1: 20) were used; all products were purchased from Chemicon, ES Cell Characterization Kit). Alkaline phosphatase staining was performed using the ES Cell Characterization Kit (Chemicon).
Cells were fixed in 4% paraformaldehyde and permeabilized with PBS containing 5% skim milk (Becton, Dickinson, USA) and 0.1% Triton X-100 for 30 min. Cells were then incubated with mouse anti-human monoclonal antibodies overnight. After washing with PBS containing 0.5% Tween 20, cells were incubated with FITC-conjugated secondary antibodies for 30 min. Negative and positive control slides were prepared by incubating sections with isotype controls instead of the primary antibody. Cells were then washed 3 times with PBS and observed by fluorescence microscopy (Olympus, Tokyo, Japan).

Ding D.C., Chu T.Y, & Liu H.W. (2018). Dedifferentiation of human uterine polyp stem cells into embryo-like cells during inducing pluripotency. International Journal of Biological Sciences, 14(11), 1586-1598.

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Characterization and Differentiation of hESCs

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The hESC colonies were cultured on chamber slides (Nunc, Thermo Fisher Scientific) in culture dishes with feeder cells, then subjected to immunohistochemistry 3–7 days following passage. Cells were fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100, blocking with 4% normal goat serum, then treatment with primary antibodies such as Sox2, stage-specific embryonic antigen-4 (SSEA4), TRA-1-60 and TRA-1-81 (ES Cell Characterization Kit; Chemicon, EMD Millipore, Billerica, MA, www.emdmillipore.com/).
For differentiation of hESC, embryoid body (EB) formation was performed for 5 days. The resulting EB was plated on gelatin-treated chamber slides and fixation. Antibodies specific for three germ layers, namely ectoderm [microtubule associated protein 2 (MAP2), tuj-1], mesoderm (brachyury) and endoderm [AT motifi-binding factor 1 (ATBF-1)], were identified.

Chang Y.H., Chu T.Y, & Ding D.C. (2017). WNT/β-Catenin signaling pathway regulates non-tumorigenesis of human embryonic stem cells co-cultured with human umbilical cord mesenchymal stem cells. Scientific Reports, 7, 41913.

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Pluripotency Marker Expression in MSCs

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Expression of Oct-4, NANOG and SOX-2 by hfC-MSCs and hBM-MSCs were studied by immunocytochemistry. The cells were fixed with 4% para-formaldehyde (Sigma Aldrich) in phosphate buffered saline (PBS), pH 7.4, for 1 hr at room temperature. The fixed cells were incubated overnight at 4°C with following primary antibodies: Oct-4, NANOG, and SOX-2 (ES Cell characterization kit; Chemicon), diluted 1:50. After washing with PBS, cells were incubated with 1:500 diluted IgG (Fab) ₂ FITC as secondary antibody (Abcam) and stained with Hoechst dye. The pictures were taken using fluorescent microscope (Nikon 80i, Japan).

Garikipati V.N., Singh S.P., Mohanram Y., Gupta A.K., Kapoor D, & Nityanand S. (2018). Isolation and characterization of mesenchymal stem cells from human fetus heart. PLoS ONE, 13(2), e0192244.

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Measuring Alkaline Phosphatase in Stem Cells

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The alkaline phosphatase (AP) activity was measured using an ES Cell Characterization kit (No. SCR001; Chemicon International, Inc., Temecula, CA), according to the manufacturer's protocol. Stained cells were imaged using an Olympus microscope (IX71; Olympus, Tokyo, Japan).

Jung J.H., Kang K.W., Kim J., Hong S.C., Park Y, & Kim B.S. (2016). CXCR2 Inhibition in Human Pluripotent Stem Cells Induces Predominant Differentiation to Mesoderm and Endoderm Through Repression of mTOR, β-Catenin, and hTERT Activities. Stem Cells and Development, 25(13), 1006-1019.

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Immunophenotyping of Pluripotent Stem Cells

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The cells were fixed with 4% paraformaldehyde, and treated with PBS containing 5% normal goat or donkey serum, 1% BSA, and 0.2% TritonX-100. The following antibodies were used: SSEA3 (1:10), TRA-1-81 (1:50), TRA-1-60 (1:50) (these antibodies were used at Kyoto University and were kind gifts from Dr. Peter W. Andrews), anti-SSEA4, anti-TRA-1-81, anti-TRA-1-60 (1:500, all contained in the ES Cell Characterization Kit from Merck Millipore; these antibodies were used at Gifu University), anti-NANOG (1:20, R&D Systems), anti-OCT3/4 (1:1000, Santa Cruz Biotechnology), anti-βIII-tubulin (1:200, Cell Signal Technology), anti-βIII-tubulin (1:2000, Covance), anti-α-SMA (1:500, DAKO), and anti-AFP (1:100, R&D). Nuclei were stained using 1 μg/mL Hoechst33342 (Life Technologies).

Tamaoki N., Takahashi K., Aoki H., Iida K., Kawaguchi T., Hatakeyama D., Inden M., Chosa N., Ishisaki A., Kunisada T., Shibata T., Goshima N., Yamanaka S, & Tezuka K.I. (2014). The homeobox gene DLX4 promotes generation of human induced pluripotent stem cells. Scientific Reports, 4, 7283.

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ES Cell Characterization Techniques

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ES cells were fixed with 4% paraformaldehyde in phosphate buffer solution (Wako, Osaka,
Japan). Alkaline phosphatase activity was detected using a commercial ES Cell
Characterization Kit (Merck Millipore), as described previously [26 (link)]. Immunostainings for SSEA1 and Oct-4 were performed using the ES
cell sample marker kit (Merck Millipore). The FITC-conjugated goat anti-mouse IgM
antibody, AP128F (Merck Millipore) was used as the secondary antibody for the detection of
SSEA1, and the Cy3-conjugated goat anti-mouse IgM antibody, AP128C (Merck Millipore) was
used as the secondary antibody for the detection of Oct-4. Stained ES cells were observed
under a fluorescence microscope (KEYENCE, Osaka, Japan).

Kumagai K., Takanashi M., Ohno S.I., Kuroda M, & Sudo K. (2016). An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice. Experimental Animals, 66(2), 125-136.

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