Staining:Article Title: Aggregate and microcarrier cultures of human pluripotent stem cells in stirred suspension systems
Article Snippet: TrypLE (Life Technologies) Cytonin (Trevigen) 2.4.3 Immunohistochemistry list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Paraformaldehyde (4%) solution Bovine serum albumin (BSA; Sigma Aldrich ) Labtek 8-well microscope glass slides with surface area 0.7 cm 2 /well (Thermo Scientific) Deionized (DI) water Octylphenylpolyethylene glycol or Triton X (J.T. .. Baker) 2.4.4 Karyotyping list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 T-75 flasks (Corning) KaryoMAX Colcemid Solution (Gibco from Life Technology) Cell hypotonic solution (CHS) is made by making a solution of 40 mM KCl, 20 mM HEPES, 0.5 mM EDTA, and 9 mM NaOH in DI water Acetic Acid Methanol GelMount aqueous mounting medium (Sigma-Aldrich) Wright-Giemsa stain, modified (Sigma-Aldrich) 2.4.5 Differentiation into mesoderm list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM (Life Technologies) Sodium selenite (Sigma Aldrich) RPMI 1640 vitamins solution (100x) (Sigma-Aldrich) Chemically defined (CD) lipid concentrate (Life Technologies; sold as 1000× solution) RPMI 1640 amino acid solution (50x) (Sigma-Aldrich) Holo-transferrin (Sigma-Aldrich) MEM non-essential amino acids (100x) (Life Technologies) Sodium pyruvate (Life Technologies; sold as 100× solution) Penicillin/streptomycin Carrier-free recombinant human BMP4 (R&D Systems) Carrier-free recombinant human WNT3A (R&D Systems) KY02111 (Xcessbio Biosciences, Inc) Matrigel or recombinant human vitronectin as substrate 2.4.6 Differentiation to ectoderm cells list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM/F12 medium Neurobasal medium (Life Technologies) B-27 supplement minus vitamin A (50x) (Life Technologies) Glutamax (100x) (Life Technologies) N2 supplement (100x) (Life Technologies) FGF2 (R&D Systems) Collagenase IV (Life Technologies) Low binding non-treated polystyrene 6-cm dishes (BD Biosciences) 2.5 Antibodies list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Anti human-NANOG (BD Pharmingen) Anti-human OCT4 (Santa Cruz Biotechnology, Inc.) Anti-human SSEA4 (Abcam) Antihuman TRA-1–60 (BD Pharmingen) Anti-human KDR (R&D Systems) Anti-human PDGFRα (R&D Systems) Anti-human C-KIT (R&D Systems) Anti-human Nestin (R&D Systems) Anti-human βIII-Tubulin (Sigma-Aldrich) Appropriate secondary antibodies (e.g. from Jackson ImmunoResearch Laboratories, Inc.). .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Human embryonic or induced pluripotent stem cells [e.g. H9 hESCs (WiCell Research Institute) or any other hPSC line of interest] Biosafety hood.
Modification:Article Title: Aggregate and microcarrier cultures of human pluripotent stem cells in stirred suspension systems
Article Snippet: TrypLE (Life Technologies) Cytonin (Trevigen) 2.4.3 Immunohistochemistry list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Paraformaldehyde (4%) solution Bovine serum albumin (BSA; Sigma Aldrich ) Labtek 8-well microscope glass slides with surface area 0.7 cm 2 /well (Thermo Scientific) Deionized (DI) water Octylphenylpolyethylene glycol or Triton X (J.T. .. Baker) 2.4.4 Karyotyping list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 T-75 flasks (Corning) KaryoMAX Colcemid Solution (Gibco from Life Technology) Cell hypotonic solution (CHS) is made by making a solution of 40 mM KCl, 20 mM HEPES, 0.5 mM EDTA, and 9 mM NaOH in DI water Acetic Acid Methanol GelMount aqueous mounting medium (Sigma-Aldrich) Wright-Giemsa stain, modified (Sigma-Aldrich) 2.4.5 Differentiation into mesoderm list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM (Life Technologies) Sodium selenite (Sigma Aldrich) RPMI 1640 vitamins solution (100x) (Sigma-Aldrich) Chemically defined (CD) lipid concentrate (Life Technologies; sold as 1000× solution) RPMI 1640 amino acid solution (50x) (Sigma-Aldrich) Holo-transferrin (Sigma-Aldrich) MEM non-essential amino acids (100x) (Life Technologies) Sodium pyruvate (Life Technologies; sold as 100× solution) Penicillin/streptomycin Carrier-free recombinant human BMP4 (R&D Systems) Carrier-free recombinant human WNT3A (R&D Systems) KY02111 (Xcessbio Biosciences, Inc) Matrigel or recombinant human vitronectin as substrate 2.4.6 Differentiation to ectoderm cells list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM/F12 medium Neurobasal medium (Life Technologies) B-27 supplement minus vitamin A (50x) (Life Technologies) Glutamax (100x) (Life Technologies) N2 supplement (100x) (Life Technologies) FGF2 (R&D Systems) Collagenase IV (Life Technologies) Low binding non-treated polystyrene 6-cm dishes (BD Biosciences) 2.5 Antibodies list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Anti human-NANOG (BD Pharmingen) Anti-human OCT4 (Santa Cruz Biotechnology, Inc.) Anti-human SSEA4 (Abcam) Antihuman TRA-1–60 (BD Pharmingen) Anti-human KDR (R&D Systems) Anti-human PDGFRα (R&D Systems) Anti-human C-KIT (R&D Systems) Anti-human Nestin (R&D Systems) Anti-human βIII-Tubulin (Sigma-Aldrich) Appropriate secondary antibodies (e.g. from Jackson ImmunoResearch Laboratories, Inc.). .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Human embryonic or induced pluripotent stem cells [e.g. H9 hESCs (WiCell Research Institute) or any other hPSC line of interest] Biosafety hood.
Recombinant:Article Title: Aggregate and microcarrier cultures of human pluripotent stem cells in stirred suspension systems
Article Snippet: TrypLE (Life Technologies) Cytonin (Trevigen) 2.4.3 Immunohistochemistry list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Paraformaldehyde (4%) solution Bovine serum albumin (BSA; Sigma Aldrich ) Labtek 8-well microscope glass slides with surface area 0.7 cm 2 /well (Thermo Scientific) Deionized (DI) water Octylphenylpolyethylene glycol or Triton X (J.T. .. Baker) 2.4.4 Karyotyping list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 T-75 flasks (Corning) KaryoMAX Colcemid Solution (Gibco from Life Technology) Cell hypotonic solution (CHS) is made by making a solution of 40 mM KCl, 20 mM HEPES, 0.5 mM EDTA, and 9 mM NaOH in DI water Acetic Acid Methanol GelMount aqueous mounting medium (Sigma-Aldrich) Wright-Giemsa stain, modified (Sigma-Aldrich) 2.4.5 Differentiation into mesoderm list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM (Life Technologies) Sodium selenite (Sigma Aldrich) RPMI 1640 vitamins solution (100x) (Sigma-Aldrich) Chemically defined (CD) lipid concentrate (Life Technologies; sold as 1000× solution) RPMI 1640 amino acid solution (50x) (Sigma-Aldrich) Holo-transferrin (Sigma-Aldrich) MEM non-essential amino acids (100x) (Life Technologies) Sodium pyruvate (Life Technologies; sold as 100× solution) Penicillin/streptomycin Carrier-free recombinant human BMP4 (R&D Systems) Carrier-free recombinant human WNT3A (R&D Systems) KY02111 (Xcessbio Biosciences, Inc) Matrigel or recombinant human vitronectin as substrate 2.4.6 Differentiation to ectoderm cells list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM/F12 medium Neurobasal medium (Life Technologies) B-27 supplement minus vitamin A (50x) (Life Technologies) Glutamax (100x) (Life Technologies) N2 supplement (100x) (Life Technologies) FGF2 (R&D Systems) Collagenase IV (Life Technologies) Low binding non-treated polystyrene 6-cm dishes (BD Biosciences) 2.5 Antibodies list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Anti human-NANOG (BD Pharmingen) Anti-human OCT4 (Santa Cruz Biotechnology, Inc.) Anti-human SSEA4 (Abcam) Antihuman TRA-1–60 (BD Pharmingen) Anti-human KDR (R&D Systems) Anti-human PDGFRα (R&D Systems) Anti-human C-KIT (R&D Systems) Anti-human Nestin (R&D Systems) Anti-human βIII-Tubulin (Sigma-Aldrich) Appropriate secondary antibodies (e.g. from Jackson ImmunoResearch Laboratories, Inc.). .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Human embryonic or induced pluripotent stem cells [e.g. H9 hESCs (WiCell Research Institute) or any other hPSC line of interest] Biosafety hood.
Binding Assay:Article Title: Aggregate and microcarrier cultures of human pluripotent stem cells in stirred suspension systems
Article Snippet: TrypLE (Life Technologies) Cytonin (Trevigen) 2.4.3 Immunohistochemistry list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Paraformaldehyde (4%) solution Bovine serum albumin (BSA; Sigma Aldrich ) Labtek 8-well microscope glass slides with surface area 0.7 cm 2 /well (Thermo Scientific) Deionized (DI) water Octylphenylpolyethylene glycol or Triton X (J.T. .. Baker) 2.4.4 Karyotyping list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 T-75 flasks (Corning) KaryoMAX Colcemid Solution (Gibco from Life Technology) Cell hypotonic solution (CHS) is made by making a solution of 40 mM KCl, 20 mM HEPES, 0.5 mM EDTA, and 9 mM NaOH in DI water Acetic Acid Methanol GelMount aqueous mounting medium (Sigma-Aldrich) Wright-Giemsa stain, modified (Sigma-Aldrich) 2.4.5 Differentiation into mesoderm list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM (Life Technologies) Sodium selenite (Sigma Aldrich) RPMI 1640 vitamins solution (100x) (Sigma-Aldrich) Chemically defined (CD) lipid concentrate (Life Technologies; sold as 1000× solution) RPMI 1640 amino acid solution (50x) (Sigma-Aldrich) Holo-transferrin (Sigma-Aldrich) MEM non-essential amino acids (100x) (Life Technologies) Sodium pyruvate (Life Technologies; sold as 100× solution) Penicillin/streptomycin Carrier-free recombinant human BMP4 (R&D Systems) Carrier-free recombinant human WNT3A (R&D Systems) KY02111 (Xcessbio Biosciences, Inc) Matrigel or recombinant human vitronectin as substrate 2.4.6 Differentiation to ectoderm cells list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 DMEM/F12 medium Neurobasal medium (Life Technologies) B-27 supplement minus vitamin A (50x) (Life Technologies) Glutamax (100x) (Life Technologies) N2 supplement (100x) (Life Technologies) FGF2 (R&D Systems) Collagenase IV (Life Technologies) Low binding non-treated polystyrene 6-cm dishes (BD Biosciences) 2.5 Antibodies list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Anti human-NANOG (BD Pharmingen) Anti-human OCT4 (Santa Cruz Biotechnology, Inc.) Anti-human SSEA4 (Abcam) Antihuman TRA-1–60 (BD Pharmingen) Anti-human KDR (R&D Systems) Anti-human PDGFRα (R&D Systems) Anti-human C-KIT (R&D Systems) Anti-human Nestin (R&D Systems) Anti-human βIII-Tubulin (Sigma-Aldrich) Appropriate secondary antibodies (e.g. from Jackson ImmunoResearch Laboratories, Inc.). .. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Human embryonic or induced pluripotent stem cells [e.g. H9 hESCs (WiCell Research Institute) or any other hPSC line of interest] Biosafety hood.
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