Human Embryonic Stem Cell Marker Panel (Hicks et al., 2020 (link); Roth et al., 2020 (link); Bomba et al., 2021 (link); Osnato et al., 2021 (link)) (Abcam, ab238602) was used to analyze pluripotency protein markers by immunofluorescence. A Trilineage Differentiation Kit (Ward et al., 2017 (link)) (STEMCELL, #05230) was used to induce differentiation into the three germ layers. Human three Germ Layer 3-Color Immunocytochemistry Kit (Han et al., 2020 (link)) (R&D, SC022) was used to identify specific protein markers of the three germ layers. A Giemsa Stain solution (Solarbio, G1015) was used to analyze the karyotype. Fluorescence images were obtained under an LSM900 laser scanning confocal microscope (Zeiss, Germany).
Human three germ layer 3 color immunocytochemistry kit
Manufactured by R&D Systems
Sourced in United States
The Human Three Germ Layer 3-Color Immunocytochemistry Kit is a laboratory tool designed for the detection and visualization of specific proteins in cells. This kit utilizes immunocytochemical techniques to label and identify cells derived from the three germ layers: ectoderm, mesoderm, and endoderm.
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Lab products found in correlation
Fluorochrome conjugated secondary antibody, by Thermo Fisher Scientific (4 mentions)
Nuclear counterstain dapi, by Merck Group (4 mentions)
Cas block, by Thermo Fisher Scientific (4 mentions)
Unconjugated primary antibodies to oct3 4, by Santa Cruz Biotechnology (4 mentions)
Tra 1 81, by Thermo Fisher Scientific (4 mentions)
A1r confocal microscope, by Nikon (4 mentions)
Psc cardiomyocyte differentiation kit, by Thermo Fisher Scientific (2 mentions)
Vectashield antifade mounting media with dapi, by Vector Laboratories (2 mentions)
Bz x810 fluorescence microscope, by Keyence (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Trilineage differentiation kit, by STEMCELL (1 mentions)
Lsm 900 confocal laser scanning microscope, by Zeiss (1 mentions)
Giemsa stain solution, by Solarbio (1 mentions)
Mouse anti tnnt2, by Thermo Fisher Scientific (1 mentions)
Rabbit anti pax6, by BioLegend (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Human cardiomyocyte immunocytochemistry kit, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Accutase, by STEMCELL (1 mentions)
11 protocols using human three germ layer 3 color immunocytochemistry kit
1
Comprehensive Stem Cell Characterization
2
Immunocytochemistry of Pluripotent Stem Cells
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The cells fixed with 4% paraformaldehyde were treated by permeabilization buffer (1% BSA and 0.1% Triton X-100) for 1 hour at room temperature. Primary antibodies against human Nanog (1:200; Cosmo Bio, Carlsbad, CA, USA) were incubated at 4°C overnight, consecutively incubation with secondary antibody (goat anti-Rabbit Texas Red-conjugated antibody; Invitrogen) was carried out. Mounted cells were examined with a fluorescence microscope or a Zeiss 510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Live staining of Tra1-81 and SSEA4 was performed using StainAlive antihuman TRA1-81, DyLight 488 and antihuman SSEA-4, DyLight 550 (Stemgent, Cambridge, MA, USA). Immunostaining for each of the three germ layers after in vitro direct differentiation from Hep3B induced pluripotent cell was performed using human three germ layer 3-color immunocytochemistry kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instruction.
3
Pluripotency and Trilineage Immunofluorescence
4
Immunostaining of Germ Layer Differentiation
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Immunostaining of three germ layers was performed using a Human Three Germ Layer 3-Color Immunocytochemistry Kit (R&D Systems). For immunostaining of cells during cardiomyocyte differentiation, the pluripotent cells were grown and differentiated in a glass-bottomed 12-well plate (MatTek, MA), and then cells were fixed and permeated in the plate using a Human Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher Scientific). The primary antibodies included mouse anti-TRA-1-60 (ESI BIO, ST11016), rabbit anti-Nanog (Stemgent, 09-0020), goat anti-Brachyury (Fisher Sciences, AF2085), rabbit anti-Pax6 (BioLegend, 901301), rabbit anti-Nkx2-5 and mouse anti-Tnnt2 (Thermo Fisher Scientific, A25973). Secondary antibodies included goat anti-mouse IgG (Alexa Fluor 488 conjugated, Thermo Fisher Scientific, A10680), donkey anti-goat IgG (DyLight 488 conjugated, Thermo Fisher Scientific, SA5-10086), goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Thermo Fisher Scientific, A-11034), and donkey anti-rabbit IgG (Alexa Fluor 594 conjugated, Thermo Fisher Scientific, A21207). The images were taken using Leica DMi8 Microsystems and Zeiss LSM710 inverted confocal microscope, and then the images were processed using the Fiji software.
5
Embryoid Body Formation and Characterization
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Embryoid bodies (EBs) cells were prepared by plating Accutase (StemCell Technologies, Vancouver, Canada) passaged iPS cells at a density of 1 to 5x104 cells per cm2 on low attachment Petri dishes for 48 hours in mTeSR. To induce differentiation, iPS cell suspensions were subsequently incubated with mTeSR supplemented with 20% FBS. EBs started to form in suspension after one week of culture. On day 20, EBs were fixed in 4% paraformaldehyde for 12 h and 70% ethanol overnight at 4°C and then embedded in paraffin, cut sections (5-7 micron) were mounted on glass slides, for immunofluorescence using Human Three Germ Layer 3-Color Immunocytochemistry Kit (R&D Systems, Minneapolis, MN).
6
Pluripotency and Trilineage Immunofluorescence
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Pluripotency immunofluorescence: iPSCs (P16-26) were fixed in 4% formaldehyde, permeabilized in 0.5% Triton® X-100 (for non-surface markers) and blocked in 3% bovine serum albumin. Cells were incubated with primary antibodies at 4 °C overnight. Cells were incubated with secondary antibodies at room temperature for one hour. Trilineage immunofluorescence: iPSCs (P10-26) were differentiated using either the StemMACS™ Trilineage Differentiation Kit, human, (Miltenyi Biotec) or to cardiac mesoderm using the PSC Cardiomyocyte Differentiation Kit (Gibco). Cells were processed for immunocytochemistry using the Human Three Germ Layer 3-Color Immunocytochemistry Kit (R&D Systems) and Alexa Fluor 488-conjugated secondary antibodies were applied where indicated. Slides were mounted in VECTASHIELD® Antifade Mounting Media with DAPI (Vector Laboratories). Cell imaging was performed using a Keyence BZ-X810 fluorescence microscope with BZ-X800 Viewer software.
7
Characterization of Genetically Modified iPSCs
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The genetically-modified iPSCs were required to ensure the normal karyotypes, the pluripotent status and the capacity to differentiate into cells of the three germ layers.
Chromosomal analysis was performed by GTG-banding analysis at the Center for Medical Diagnostic Laboratories, Faculty of Medicine, Chulalongkorn University, Thailand, following the recommendations by the International System for Cytogenetics Nomenclature (ISCN).
The pluripotency of stem cells was evaluated by reverse-transcriptase PCR (RT-PCR) of stem cell markers including NANOG, OCT4, SOX2, KLF4 and MYC. The primer sequences are listed in Table1 31 (link)–34 (link).
The ability of stem cells to differentiate into endoderm, mesoderm and ectoderm were tested via embryoid body (EB) formation and stained by Human three germ layer 3-color immunocytochemistry kit (R&D Systems, Minneapolis, MN, USA)35 (link).
To demonstrate the hematopoietic multipotency, our iPSC-derived hematopoietic progenitors were cultured in the presence of 100 ng/ml human thrombopoietin (TPO), 50 ng/ml human stem cell factor (SCF), and 25 ng/ml heparin for 21 days. The culture finally yielded the mixture of megakaryocytes (by morphology and flow cytometry for CD41/CD42b surface expression) and neutrophils (by morphology) as shown in the Supplementary Fig.S8 .
Chromosomal analysis was performed by GTG-banding analysis at the Center for Medical Diagnostic Laboratories, Faculty of Medicine, Chulalongkorn University, Thailand, following the recommendations by the International System for Cytogenetics Nomenclature (ISCN).
The pluripotency of stem cells was evaluated by reverse-transcriptase PCR (RT-PCR) of stem cell markers including NANOG, OCT4, SOX2, KLF4 and MYC. The primer sequences are listed in Table
The ability of stem cells to differentiate into endoderm, mesoderm and ectoderm were tested via embryoid body (EB) formation and stained by Human three germ layer 3-color immunocytochemistry kit (R&D Systems, Minneapolis, MN, USA)35 (link).
To demonstrate the hematopoietic multipotency, our iPSC-derived hematopoietic progenitors were cultured in the presence of 100 ng/ml human thrombopoietin (TPO), 50 ng/ml human stem cell factor (SCF), and 25 ng/ml heparin for 21 days. The culture finally yielded the mixture of megakaryocytes (by morphology and flow cytometry for CD41/CD42b surface expression) and neutrophils (by morphology) as shown in the Supplementary Fig.
8
Immunocytochemical Characterization of Pluripotent Stem Cells
9
Immunocytochemical Analysis of Pluripotency and Lineage Markers
10
Immunocytochemical Analysis of Pluripotency and Lineage Markers
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Cells grown on matrigel-coated chamber slides were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, and incubated with CAS-Block™ (Thermo Fischer Scientific Inc., Waltham, MA) for 30 min. Unconjugated primary antibodies to Oct3/4 (Santa Cruz Biotechnology, Dallas, Texas) and TRA-1-81 (Thermo Fischer Scientific Inc.), or fluorochrome conjugated antibodies to SOX1, Otx-2, Brachyury, and GATA4 (Human Three Germ Layer 3-Color Immunocytochemistry Kit, R&D Systems Inc., Minneapolis, MN) were incubated overnight at 4 °C. When necessary, cells were incubated with fluorochrome conjugated secondary antibodies (Thermo Fischer Scientific Inc.) for 4 h at room temperature under humid conditions. Coverslips were mounted with fluoroshield mounting medium containing nuclear counterstain DAPI (Sigma Aldrich, St. Louis, MO). Cells were visualized with confocal imaging (Nikon A1-R confocal microscope; Nikon Instruments Inc., Melville, NY).
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