For the hepatic differentiation of human PSCs, a previously reported hepatic differentiation protocol designed for human ESCs [1 (link)] was applied with some modifications. Briefly, human PSCs were plated at a density of 2.5 × 105 cells/ml on Matrigel-coated six-well plates with mTeSR1 medium (STEMCELL Technologies) including ROCK inhibitor (Y27632; STEMCELL Technologies). The medium was replaced with definitive endodermal induction medium (DE) for 5 days (stage I). The DE medium consisted of RPMI 1640 (without l -glutamine; Gibco-BRL) supplemented with 2 mM l -glutamine (Millipore), 0.5 mg/ml albumin fraction V (Merck Millipore, Germany) and 100 ng/ml Activin A (PeproTech, NJ, USA). Afterward, the definitive endodermal cells were differentiated into hepatoblasts using the hepatic endodermal medium (Hep-1) for 5 days (stage II) followed by hepatic specification medium (Hep-2) for 5 days (stage III). Hep-1 and Hep-2 comprised the HBM SingleQuots™ kit (Lonza) supplemented with 30 ng/ml FGF4 (PeproTech) and 20 ng/ml BMP2 (Invitrogen, MD, USA) at stage II and 20 ng/ml HGF (PeproTech) and 20 ng/ml OSM (R&D Systems, MN, USA) at stage III, respectively. After hepatic specification, the cells were further matured using the HMM SingleQuots™ kit for 5 days (stage IV).
Albumin fraction 5
Manufactured by Merck Group
Sourced in Germany
Albumin Fraction V is a laboratory product manufactured by Merck Group. It is a purified form of bovine serum albumin, a common protein used in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Hexahydrate, by Merck Group (3 mentions)
γ al 2 o 3 nanoparticles, by IoLiTec (3 mentions)
Activin a, by Thermo Fisher Scientific (2 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 without l glutamine, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Methanol p a, by Merck Group (1 mentions)
Ethanol p a, by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
Silica gel 60 f254, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Bmp 2, by Thermo Fisher Scientific (1 mentions)
Oncostatin m, by R&D Systems (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Lsm710 system, by Zeiss (1 mentions)
Nb600 308, by Santa Cruz Biotechnology (1 mentions)
Lsm 710, by Zeiss (1 mentions)
8 protocols using albumin fraction 5
1
Hepatic Differentiation of Human PSCs
2
Kencur Rhizome Extraction and Analysis
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Materials used during the study included kencur rhizome, ethanol p.a. (Merck KGaA), methanol p.a. (Merck KGaA), ethyl acetate p.a. (Merck KGaA), Silica Gel 60 F 254 (Merck KGaA), ethyl pmethoxycinnamate (Santa Cruz Biotechnology), NaCl (Merck KGaA), Tris Base (Merck KGaA), Albumin fraction V (Merck KGaA), Aquadest (Bratachem).
3
Oridonin Anti-Cancer Mechanism Study
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Oridonin (CAS no. 28957-04-2) was acquired from Aladdin Shanghai Biochemical Technology Co., Ltd. (Shanghai, China). MTT, dimethyl sulfoxide (DMSO) and albumin fraction V (BSA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Thermo Fisher Scientific Inc., Waltham, MA, USA). Antibodies against E-cadherin (mouse; cat. no. 14472), N-cadherin (rabbit; cat. no. 13116), Vimentin (mouse; cat. no. 3390) and Snail (mouse; cat. no. 3895) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against HIF-1α (mouse; cat. no. ab113642), VEGF-A (rabbit; cat. no. ab46154), VEGFR-2 (rabbit; cat. no. ab2349), β-actin (rabbit; cat. no. ab8227) and CD31 (rabbit; cat. no. ab32457) were purchased from Abcam (Cambridge, MA, USA). Goat anti-mouse IgG-horseradish peroxidase (HRP) (cat. no. sc-2005) and goat anti-rabbit IgG-HRP (cat. no. sc-2004) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
4
Hepatocyte Differentiation from hESCs
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hESCs were differentiated into hepatocytes as previously described (19 (link)). Briefly, for definitive endoderm (DE) induction, hESCs were cultured in RPMI1640 medium (Hyclone, UT) supplemented with 0.5 mg/ml albumin fraction V (Sigma-Aldrich) and 50 ng/ml Activin A (Peprotech, NJ, USA) for 1 d and 1% insulin-transferrin-selenium (ITS, Sigma-Aldrich) was added to this medium for an additional 4 d. For hepatocyte differentiation, hESC-derived DE cells were cultured in hepatocyte culture medium (HCM, Lonza, MD) containing 30 ng/ml FGF4 (Peprotech) and 20 ng/ml BMP2 (Peprotech) for 5 d, then incubated in HCM containing 20 ng/ml HGF (Peprotech) for 5 d, and further cultured in HCM supplemented with 10 ng/ml Oncostatin M (OSM, R&D systems) and 0.1 μM DEXamethasone (DEX, Sigma-Aldrich) for 5 d. The medium was changed daily.
5
Whole-Mount Immunostaining of Embryos
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In situ hybridization and whole-mount staining were performed as described in ref. 18 (link). Briefly, for whole-mount immunostaining embryos were fixed with 2% formaldehyde in 0.1 M PIPES (Sigma), 1 mM MgSO4 and 2 mM EGTA overnight at 4 °C (pH of the solution 7.4). Embryos were washed with PBS and blocked for 1 h in PBS with 5% BSA (Albumin fraction V, Sigma) and 0.3% Triton X-100 at 37 °C. The embryos were then incubated with primary antibodies diluted in blocking solution (α-Isl1/2 supernatant 1:10 (DSHB, 39.4D5); α-GFP 1:500 (Novus Biologicals, NB600-308); α-MEF-2 (C-21) 1:250 (Santa Cruz, sc-313); α-MHC supernatant 1:10 (DSHB, MF20); α-MYH6 supernatant 1:10 (DSHB, S46)) overnight at 4 °C. Embryos were washed three times with 0.3% Triton X-100 in PBS for 1 h at 4 °C and incubated with secondary antibodies diluted in blocking solution (Alexa conjugates 1:500) overnight at 4 °C. Final washes were done with 0.3% Triton X-100 in PBS, three times for 1 h at 4 °C. For sectioning, embryos were embedded in 17% gelatin in PBS. The gelatin cubes were fixed overnight at room temperature in 4% PFA in PBS and sections were performed with a Vibratome VT 1000 S (Leica). Confocal images were acquired on a Zeiss LSM 710 system and Z-stacks projections were generated using Zeiss LSM 710 software.
6
Nanomaterial Preparation and Characterization
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Most chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), or Carl Roth (Karlsruhe, Germany) in the highest available purity.
Nanomaterials (Al 0 -core surface-passivated nanoparticles and γ-Al 2 O 3 nanoparticles) were supplied by IoLiTec (Heilbronn, Germany). Al 0 nanoparticles were stored and weighted under an argon atmosphere. Both particle species were freshly dispersed according to the modified NanoGenoTOX protocol (dispersion in 0.05% BSA/water by ultrasonication with KE76 tip for 5'09'' with ~20% energy), BSA was supplied by Carl Roth (Albumin Fraction V, ≥98%)
and AlCl 3 was supplied by Sigma-Aldrich (Hexahydrate, ≥97%).
Nanomaterials (Al 0 -core surface-passivated nanoparticles and γ-Al 2 O 3 nanoparticles) were supplied by IoLiTec (Heilbronn, Germany). Al 0 nanoparticles were stored and weighted under an argon atmosphere. Both particle species were freshly dispersed according to the modified NanoGenoTOX protocol (dispersion in 0.05% BSA/water by ultrasonication with KE76 tip for 5'09'' with ~20% energy), BSA was supplied by Carl Roth (Albumin Fraction V, ≥98%)
and AlCl 3 was supplied by Sigma-Aldrich (Hexahydrate, ≥97%).
7
Nanomaterial Preparation and Stabilization
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Standard chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), or Carl Roth (Karlsruhe, Germany) in the highest available purity.
Nanomaterials (Al 0 -core surface-passivated nanoparticles and γ-Al2O3 nanoparticles) were supplied by IoLiTec (Heilbronn, Germany). Al 0 nanoparticles were stored and weighed under an argon atmosphere. Both particles were freshly dispersed according to the modified NanoGenoTOX protocol (ultrasonication with KE76 for 5'09'' with ~20% energy), stabilized by 0.05% BSA/water before use (Krause et al., 2018) . BSA was supplied by Carl Roth (Albumin Fraction V, ≥98%) and AlCl3 was supplied by Sigma-Aldrich (Hexahydrate, ≥97%).
Nanomaterials (Al 0 -core surface-passivated nanoparticles and γ-Al2O3 nanoparticles) were supplied by IoLiTec (Heilbronn, Germany). Al 0 nanoparticles were stored and weighed under an argon atmosphere. Both particles were freshly dispersed according to the modified NanoGenoTOX protocol (ultrasonication with KE76 for 5'09'' with ~20% energy), stabilized by 0.05% BSA/water before use (Krause et al., 2018) . BSA was supplied by Carl Roth (Albumin Fraction V, ≥98%) and AlCl3 was supplied by Sigma-Aldrich (Hexahydrate, ≥97%).
8
Synthesis and Characterization of Aluminum Nanoparticles
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Chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), or Carl Roth (Karlsruhe, Germany) in the highest available purity. Nanomaterials (Al 0 -core surface-passivated nanoparticles and γ-Al 2 O 3 nanoparticles) were supplied by IoLiTec. Al 0 nanoparticles were stored and weighted under an argon atmosphere. Both particles were freshly dispersed at a concentration of 2.56 mg/ml according to the modified NanoGenoTOX protocol (ultrasonication applying an energy of 1176 kJ/ml dispersion using an acoustic power of 7.35 W), stabilized by 0.05% BSA/water before use. BSA was supplied by Carl Roth (Albumin Fraction V, ≥98%) and AlCl 3 was supplied by Sigma Aldrich (Hexahydrate, ≥97%).
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