Ssea 1

Manufactured by Merck Group

Sourced in United States

SSEA-1 is a laboratory reagent that is used to detect the presence of the SSEA-1 (stage-specific embryonic antigen-1) marker. SSEA-1 is a carbohydrate antigen expressed on the surface of undifferentiated embryonic stem cells and early stage embryos. This reagent can be used in various cell biology and developmental biology research applications.

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Lab products found in correlation

Ssea 4, by Merck Group (8 mentions) Triton x 100, by Merck Group (5 mentions) Tra 1 60, by Merck Group (4 mentions) Nanog, by Abcam (3 mentions) Nanog, by Merck Group (3 mentions) Nanog, by Cosmo Bio (2 mentions) Lsm 510 meta, by Zeiss (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (2 mentions) Rabbit anti zo 1 antibody, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde solution, by Electron Microscopy Sciences (2 mentions) Oct 4, by Merck Group (2 mentions) Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions) Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Lsm 700 laser scanning microscope, by Zeiss (2 mentions) Zen 2012 light edition program, by Zeiss (2 mentions) Ssea3, by Merck Group (2 mentions) Chamber slide, by Thermo Fisher Scientific (1 mentions) Dapi mounting medium, by Vector Laboratories (1 mentions)

17 protocols using ssea 1

Induction of Pluripotency in Stem Cells

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Two HUESC lines, that is, HUES-24 and HUES-9 pOct4/green fluorescent protein (GFP), were used throughout this study without any difference in the data. HUESCs were cultured on mouse embryonic fibroblasts from E14 Mouse embryos, using KO-DMEM medium supplemented with β-mercaptoethanol, glutamine, non-essential amino acids, 15% knock-out serum replacement (KOSR) and 10 ng ml⁻¹ fibroblast growth factor 2 (FGF2). Medium was changed every day. Cell colonies were dissociated into single cells every 4 days using trypsin. Cell nucleofection with OCT4-iA was performed with AMAXA kit according to the manufacturer. The cells were then plated on matrigel-coated dishes in HUESCs MEF-conditioned medium supplemented with FGF2 for 4 days to reach a twofold increase in the OCT4 protein level.
Cells were phenotyped every 10 passages using anti-SSEA-3/4, TRA-1–60 and TRA-1–80 antibodies used at 1/100 (Chemicon kit SCR-002). Less than 5% of cells were positive for SSEA-1 (Chemicon). Karyotype was found normal and stable in the course of the experiments.
EBs were generated from mock pcDNA nucleofected or Oct4-iA-nucleofected HUESCs 4 days post nucleofection in DMEM added with 20% FCS. Cells were let in suspension for 6 days to allow for their aggregation and then EBs were dissociated with trypsin and plated on cells plated for 14 h on fibronectin-coated labtech plates.

Abboud N., Moore-Morris T., Hiriart E., Yang H., Bezerra H., Gualazzi M.G., Stefanovic S., Guénantin A.C., Evans S.M, & Pucéat M. (2015). A cohesin–OCT4 complex mediates Sox enhancers to prime an early embryonic lineage. Nature communications, 6, 6749.

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Immunophenotyping of Human iPSCs

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AP staining was performed using the Alkaline Phosphatase Detection Kit (Sigma) according to the manufacturer’s instructions. For immunofluorescence assay, human iPSCs were adhered on slide chambers (Nunc) containing MEFs, fixed with 4% paraformaldehyde (Sigma) for 30 minutes at RT and permeabilized for 10 minutes with 1% TritonX-100 (Sigma). Cells were blocked with 5% BSA for 30 minutes at RT and incubated for one hour at RT or o/n at 4°C with NANOG antibody (Abcam), SOX2 (R&D), SSEA-1 (Chemicon), SSEA-4 (Chemicon), TRA1-60 (Chemicon) and TRA1-81 (Chemicon) diluted in PBS/TBS with 1% BSA. FITC- or Cy3-conjugated secondary antibodies (Sigma) diluted in the same solution was incubated for 1–1.5 hours at RT. Nuclei were counterstained with 1:4 dilution of DAPI mounting medium (Vector Labs). Samples were visualized under an inverted fluorescence microscope.

Rodriguez-Madoz J.R., San Jose-Eneriz E., Rabal O., Zapata-Linares N., Miranda E., Rodriguez S., Porciuncula A., Vilas-Zornoza A., Garate L., Segura V., Guruceaga E., Agirre X., Oyarzabal J, & Prosper F. (2017). Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome. PLoS ONE, 12(12), e0190275.

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Immunophenotyping of Stem Cell Colonies

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AP staining was performed using the Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich) following protocols provided by the manufacturer. For immunofluorescence, colonies were fixed for 2 h at room temperature with 4% paraformaldehyde and then incubated at room temperature for 15 min with 1% Triton X-100/phosphate buffer (PBS). Cells were washed three times in PBS and blocked at 37 °C for over 3 h with 4% normal goat serum (Chemicon). Subsequently, cells were incubated at 4 °C overnight with primary antibody against Oct4 (1:500, Santa Cruz Biotechnology), SSEA-1 (1:500, Chemicon), Nanog (1:500, Cosmobio), or Sox2 (1:500, Abcam). Cells were washed three times in PBS and incubated at 37 °C for 2 h with goat anti-rabbit Alexa-Flour 594-conjugated (Life Technologies) and goat anti-mouse Alexa-Fluor IgG or IgM 633-conjugated (Molecular Probes) secondary antibodies (1:500 in 1% normal goat serum in PBS). Unbound secondary antibody was removed using three washes with PBS. Nuclei were identified by DAPI (Invitrogen) staining at a dilution of 1:1,000,000 at room temperature for 5 min. Images were acquired using a confocal laser scanning microscope (LSM 510 META, Carl Zeiss).

Miao X., Li Y., Zheng C., Wang L., Jin C., Chen L., Mi S., Zhai W., Wang Q.F, & Cai J. (2020). A promising iPS-based single-cell cloning strategy revealing signatures of somatic mutations in heterogeneous normal cells. Computational and Structural Biotechnology Journal, 18, 2326-2335.

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Immunofluorescence Staining and Imaging Protocol

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Cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 min. The cells were then permeabilized with 0.2% Triton X-100, and non-specific binding sites were blocked with Block Ace (Yukijirushi). Cells were incubated with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 1 hr. Fluorescent images were captured using IN Cell Analyzer 6000, and the percentage of MNs or neurons was calculated using IN Cell Developer Toolbox v1.9 (GE Healthcare). For phenotype assays, images were acquired by Delta Vision (GE Healthcare). The primary antibodies were as follows: HB9 (Developmental Studies Hybridoma Bank [DSHB], 1:200), Tuj1 (Covance, 1:2,000), Tuj1 (Chemicon, 1:500) for Figures 6A and 6C, ChAT (Chemicon, 1:100), HOXB4 (DSHB, 1:50), HOXC6 (Abcam, 1:200), HOXC9 (Abcam, 1:200), HOXC10 (Abcam, 1:2,000), misfolded SOD1 (MEDIMABS, B8H10, 1:200), misfolded SOD1 (MEDIMABS, A5C3, 1:200), TDP-43 (Proteintech, 1:200), human Nanog (ReproCELL, 1:500), SSEA4 (Millipore, 1:200), SSEA1 (Chemicon, 1:1,000), Nestin (Millipore, 1:200), GFAP (Dako, 1:2,000), Iba1 (Wako Pure Chemicals Industries, 1:500), CNPase (Cell Signaling Technology, 1:100), SOX17 (R&D Systems, 1:200), and αSMA (Dako, 1:500). Tuj1 (Chemicon) was used to co-immunostain with TDP-43.

Goto K., Imamura K., Komatsu K., Mitani K., Aiba K., Nakatsuji N., Inoue M., Kawata A., Yamashita H., Takahashi R, & Inoue H. (2017). Simple Derivation of Spinal Motor Neurons from ESCs/iPSCs Using Sendai Virus Vectors. Molecular Therapy. Methods & Clinical Development, 4, 115-125.

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Immunofluorescence Staining of Stem Cells

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For immunofluorescence staining, AN and AI cells were seeded on gelatin-coated cover slips and fixed with 4% paraformaldehyde. After permeabilization with 0.5% Triton-X and blocking with 0.5% bovine serum albumin (BSA), the cells were incubated with primary antibodies against Oct4 (1:500, Santa Cruz, Dallas, TX, USA), Sox2 (1:500, Santa Cruz), Nanog (1:500, COSMO BioCo, Tokyo, Japan) and SSEA-1 (1:50, Merck, Millipore). Then, the cells were incubated with the appropriate secondary antibodies after washing three times. DNA was labeled with DAPI (Merck, Millipore). Stained cells were mounted on cover slips and observed using a LSM 510 META microscope (Zeiss, North York, ON, Canada).

Li H., Gao S., Huang H., Liu W., Huang H., Liu X., Gao Y., Le R., Kou X., Zhao Y., Kou Z., Li J., Wang H., Zhang Y., Wang H., Cai T., Sun Q., Gao S, & Han Z. (2017). High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells. Oncotarget, 8(29), 47344-47355.

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Immunofluorescence Staining of Stem Cells

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Cells were washed once in PBS, and fixed in 4% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, PA) for 15 min. Cells were washed three times in PBS at room temperature and permeabilized by exposure to 1% Triton X-100 (Sigma, St. Lois, MO) for 1 min at room temperature. Samples were washed three times in PBS at room temperature and blocked with 1% goat serum for 45 min followed by incubation with primary antibodies (TRA-1-60, 1:50, SSEA4, 1:50, SSEA1, 1:50, OCT-4, 1; 50, EMD Millipore, MA), RPE65 antibody (1:250) (Golczak et al., 2010 (link)) and rabbit anti-ZO-1 antibody (1:200, Invitrogen) for 1 h at room temperature. The cells were washed three times in PBS, and incubated with secondary antibodies (Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 555 goat anti-mouse IgG (1:500; Invitrogen) for 30 min at room temperature followed by PBS wash three times for 5 min. DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes, Eugene, OR) was used to stain nuclei. Cells were visualized and imaged under an inverted fluorescence microscope.

Parmar V.M., Parmar T., Arai E., Perusek L, & Maeda A. (2018). A2E-associated cell death and inflammation in retinal pigmented epithelial cells from human induced pluripotent stem cells. Stem cell research, 27, 95-104.

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Immunofluorescence Staining of Pluripotent Stem Cells

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For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde, blocked using blocking solution (3% BSA and 2% serum of host secondary antibodies with 0.1% Tween-20 in PBS), and incubated with primary antibodies overnight. Then, the cells were washed with 0.1% Tween-20 in PBS and incubated with fluorescent-conjugated secondary antibodies. After washing, the cell nuclei were counterstained with DAPI. The antibodies used in the study were; SSEA-1 (Merck Millipore, Burlington, MA, USA), SOX2 (Merck Millipore, Burlington, MA, USA), Oct-3/4 (Santa Cruz, Dallas, TX, USA), NANOG (Abcam, Cambridge, UK), donkey anti-rabbit or anti-goat IgG conjugated with Alexa Fluor 488, and anti-rabbit conjugated with Alexa Fluor 546 (Thermo Fisher Scientific, Carlsbad, CA, USA). For AP staining of the ESCs, the samples were fixed and stained using an Alkaline Phosphatase Staining Kit II (Stemgent, Beltsville, MD, USA) following the manufacturer’s instructions.

Yoo H., La H., Lee E.J., Choi H.J., Oh J., Thang N.X, & Hong K. (2020). ATP-Dependent Chromatin Remodeler CHD9 Controls the Proliferation of Embryonic Stem Cells in a Cell Culture Condition-Dependent Manner. Biology, 9(12), 428.

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Immunofluorescence Staining of Stem Cells

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Protein Detection and Immunoprecipitation

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Cells were lysed with lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 12.5 mM b-glycerophosphate, 1.5 mM MgCl2, 2 mM ethylene glycol tetraacetic acid, 10 mM NaF, and 1 mM Na3VO4) containing protease inhibitors (Roche). Western blot was performed by standard procedures; primary antibodies used in this study: anti-Oct4 (Santa Cruz, sc-365509), anti-Rbm46 (sigma, HPA050601), beta-Actin (santa cruz, sc-47778), Pabpc1 (AVIVA, OAAB01699), GSK-3 (Millipore, 05–412), SSEA-1(Millipore, FCMAB117P). Proteins were visualized with an Odyssey Two-Color Infrared Imaging System (LI-COR Biosciences) according to the manufacturer’s instructions. For Flag immunoprecipitaion, Flag M2 beads (Sigma, F1804) were utilized according to the manufacturer’s protocal.

Zhai L., Wang C., Chen Y., Zhou S, & Li L. (2017). Rbm46 regulates mouse embryonic stem cell differentiation by targeting β-Catenin mRNA for degradation. PLoS ONE, 12(2), e0172420.

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Pluripotency and Differentiation Gene Expression Analysis

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ICC analyses were performed to evaluate the expression of genes related to pluripotency and differentiation. Before staining, all cell samples were preincubated for 10 min at 4°C and fixed with 4% paraformaldehyde for 30 min. After washing twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene), samples were treated for 1 h with 10% goat serum in DPBS to prevent nonspecific binding. Serum-treated cells were incubated overnight at 4°C with primary antibodies. The primary antibodies used were as follows: OCT4 (Santa Cruz Biotechnology, CA, USA; 1:200), SOX2 (Millipore; 1:200), NANOG (Santa Cruz Biotechnology; 1:200), SSEA1 (Millipore; 1:200), SSEA4 (Millipore; 1:200), Neurofilament (Millipore; 1:100), Vimentin (Millipore; 1:100), and Cytokeratin 17 (Millipore; 1:100). When we used the antibodies for intracellular proteins such as OCT4, SOX2, and NANOG, fixed cells were treated for 5 min with 0.2% Triton-X100 (Sigma-Aldrich, MO, USA) before serum blocking. After incubation with the primary antibody, the cells were treated for 3 h at room temperature with Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with Hoescht 33342 (Molecular Probes). Images of stained cells were captured using a LSM 700 Laser Scanning Microscope (Carl Zeiss, Germany) and processed with the ZEN 2012 Light Edition program (Carl Zeiss).

Choi K.H., Park J.K., Son D., Hwang J.Y., Lee D.K., Ka H., Park J, & Lee C.K. (2016). Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig. PLoS ONE, 11(6), e0158046.

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