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3 germ layer immunocytochemistry kit

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The 3-Germ Layer Immunocytochemistry Kit is a laboratory tool designed for the identification and analysis of cells derived from the three primary germ layers: ectoderm, mesoderm, and endoderm. The kit provides a set of specific antibodies and reagents to perform immunocytochemical staining and detection of these cell lineages.

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Lab products found in correlation

V bottomed conical wells, by Sumitomo Bakelite (2 mentions) Tryple express, by Thermo Fisher Scientific (2 mentions) Collagenase type 4, by Thermo Fisher Scientific (1 mentions) Polyvinyl alcohol (pva), by Merck Group (1 mentions) Ultra low attachment 6 well plate, by Corning (1 mentions) Tcs sp2 microscope, by Leica (1 mentions) Tcs sp8 sted 3x confocal microscope, by Leica (1 mentions) Matrigel, by Corning (1 mentions) 6 well plate, by Sarstedt (1 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) Psc 4 marker immunocytochemistry kit, by Thermo Fisher Scientific (1 mentions)

10 protocols using 3 germ layer immunocytochemistry kit

1

Differentiation and Characterization of Embryoid Bodies

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Embryoid bodies (EBs) were generated after passaging of iPSC and ESC colonies using type
IV collagenase (1 mg/mL) (Life Technologies, Carlsbad, USA) and cultured in suspension
culture on Petri dishes. Differentiation medium consisted of Essential 8 supplemented
with
4 mg/mL polyvinyl alcohol (PVA) (Sigma-Aldrich, St. Louis, USA) to prevent colony
adhesion. After 5 days of incubation, EBs were transferred onto cover glasses and placed
in adhesive culture dishes. They were fixed after 14 days according to the abovementioned
protocol for immunofluorescence staining.
EBs were immunostained against derivatives of the three germ layers, AFP, SMA, and TUJ1,
using a 3-Germ Layer Immunocytochemistry Kit (Thermo Fisher Scientific, Carlsbad, USA)
according to the manufacturer’s instructions.
Lewandowski J., Rozwadowska N., Kolanowski T.J., Malcher A., Zimna A., Rugowska A., Fiedorowicz K., Łabędź W., Kubaszewski Ł., Chojnacka K., Bednarek-Rajewska K., Majewski P, & Kurpisz M. (2018). The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin. Cell Transplantation, 27(7), 1047-1067.
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2

Pluripotency Validation of Human iPSCs

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Embryoid body formation was used to confirm differentiation potential into the three germ layers in vitro. Human iPSCs were cultured in DMEM/F12 supplemented with 20% FBS in ultra-low attachment 6-well plates (Corning) for 7 days after which embryoid bodies were re-plated onto 0.1% gelatin-coated plates. Fourteen days after re-plating, cells were then fixed with 4% formaldehyde and stained with antibodies against ectodermal marker βIII-Tubulin (TUJI), mesodermal marker α-SMA, and endodermal marker, AFP (3-Germ Layer Immunocytochemistry Kit, Thermo Fisher Scientific).
Huang C.Y., Li L.H., Hsu W.T., Cheng Y.C., Nicholson M.W., Liu C.L., Ting C.Y., Ko H.W., Syu S.H., Wen C.H., Yan Z., Huang H.P., Su H.L., Chiang P.M., Shen C.N., Chen H.F., Yen B.L., Lu H.E., Hwang S.M., Chiou S.H., Ho H.N., Wu J.Y., Kamp T.J., Wu J.C, & Hsieh P.C. (2020). Copy number variant hotspots in Han Taiwanese population induced pluripotent stem cell lines - lessons from establishing the Taiwan human disease iPSC Consortium Bank. Journal of Biomedical Science, 27, 92.
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3

Directed Differentiation of Embryonic Bodies

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Embryonic bodies were generated by following the instructions of the AggreWell™800 Starter Kit (cat# 34850, STEMCELL Technologies). Briefly, cells were harvested with the Gentle Cell Dissociation Reagent and 1.2 million cells were plated in the AggreWell™800 plates and incubated for 24 hours. The generation of embryonic bodies was facilitated by culturing the embryonic bodies in Primate ES Cell Media (cat# 258RCHEMD001, Tebu Bio), the first 10 days in suspension plates (cat# 83.3920.500, Sarstedt) followed by 14 days in 6-well plates (cat# 83.3920, Sarstedt), coated with Matrigel (cat# 354230, Corning). The embryonic bodies were stained for beta-III tubulin (TUJ1), smooth muscle actin (SMA), and alpha-fetoprotein (AFP) by following the instructions of the 3-germ layer immunocytochemistry kit (cat# A25538, Thermo). The expression of TUJ1 (1/500), SMA (1 : 100), and AFP (1 : 500) were analyzed by using a Leica TCS SP2 microscope with a 40x objective, a Leica TCS SP5 confocal with a 40x objective, or a Leica TCS SP8 STED 3X confocal microscope with a 100x objective.
Bjørlykke Y., Søviknes A.M., Hoareau L., Vethe H., Mathisen A.F., Chera S., Vaudel M., Ghila L.M, & Ræder H. (2019). Reprogrammed Cells Display Distinct Proteomic Signatures Associated with Colony Morphology Variability. Stem Cells International, 2019, 8036035.
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4

Immunostaining of Embryoid Bodies

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EBs were derived from iPSCs and subcultured in the EB medium for 2 weeks. Mature EBs were collected for immunostaining of three germ layers markers, and the staining was performed according to the manufacturer’s instruction of 3-Germ Layer Immunocytochemistry Kit (Thermo Fisher Scientific). In detail, EBs were first fixed and permeabilized at room temperature. Before the staining, EBs were also incubated with a blocking solution for 30 min. After the blocking, primary antibodies were added to stain EBs at 4 °C overnight. Subjected to washing, EBs were then stained with secondary antibodies at room temperature for 1 h. After the second round of washing, stained EBs were visualized by a microscope.
Zha S., Tay J.C., Zhu S., Li Z., Du Z, & Wang S. (2020). Generation of Mesenchymal Stromal Cells with Low Immunogenicity from Human PBMC-Derived β2 Microglobulin Knockout Induced Pluripotent Stem Cells. Cell Transplantation, 29, 0963689720965529.
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5

Pluripotent Stem Cell Analysis

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For immunofluorescence analysis, the PSC 4-marker Immunocytochemistry Kit (A24881; Thermo Fisher Scientific) and the 3-Germ Layer Immunocytochemistry Kit (A25538; Thermo Fisher Scientific) coupled with NCAM antibody (MA1–06,801; Thermo Fisher Scientific; RRID: AB_558,237) were used, following manufacturer's instructions. Fluorescence mounting medium (DakoCytomation, Glostrup, Denmark) was used. Samples were imaged on a Nikon Eclipse 80i VideoConfocal microscope (Nikon).
Barilani M., Cherubini A., Peli V., Polveraccio F., Bollati V., Guffanti F., Del Gobbo A., Lavazza C., Giovanelli S., Elvassore N, & Lazzari L. (2020). A circular RNA map for human induced pluripotent stem cells of foetal origin. EBioMedicine, 57, 102848.
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6

Germ Layer Differentiation Assay

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Important property of stem cells is their ability to differentiate to cells specific for the three germ layers. Therefore, the 3-germ layer immunocytochemistry kit (Thermo Fisher Scientific Inc.) was used. The procedure applied herein enables the detection of relevant germ layers markers, such as beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and Smooth Muscle Actin (SMA) for mesoderm. The procedure has been performed according to the manufacturer’s instructions. Pictures were taken under inverted light microscope with fluorescence (OLYMPUS, Japan).
Fus-Kujawa A., Mendrek B., Bajdak-Rusinek K., Diak N., Strzelec K., Gutmajster E., Janelt K., Kowalczuk A., Trybus A., Rozwadowska P., Wojakowski W., Gawron K, & Sieroń A.L. (2023). Gene-repaired iPS cells as novel approach for patient with osteogenesis imperfecta. Frontiers in Bioengineering and Biotechnology, 11, 1205122.
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7

Multilineage Differentiation Tracking of hESCs

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The HBB-Cl11-GFP line was treated with 0.5 mM EDTA in 1X PBS. Cells were gently scraped with a cell scraper to form small aggregates (10–20 cells). Aggregates were resuspended in hESC media in the absence of bFGF and in the presence of thiazovivin (2 μM). The EBs were cultured in this media for 5 days and the EBs allowed to settle in a falcon tube. The hESC media was aspirated off and resuspended in DMEM containing 10% FBS and replated on Matrigel coated plates and EB cells were allowed to attach. Media was changed every 3 days and the culture was maintained for 14 days. Random differentiation was detected by simultaneous 3-color staining using three fluorochrome-conjugated antibodies provided with the 3 Germ Layer Immunocytochemistry Kit (Invitrogen; Catalog # A25538). Ectoderm derivatives were detected with TUJ1-rabbit (AF647), mesoderm differentiated cells with SMA-mouse (AF555), and endoderm derivatives with AFP-mouse IgG1 (AF488). The nuclei were counterstained with DAPI (blue).
Alexeeva V., Aydin I.T., Schaniel C., Stranahan A.W., D’Souza S.L, & Bieker J.J. (2020). A human H1-HBB11-GFP reporter embryonic stem cell line (WAe001-A-2) generated using TALEN-based genome editing. Stem cell research, 45, 101837.
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8

Multilineage Differentiation Tracking of hESCs

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The HBB-Cl11-GFP line was treated with 0.5 mM EDTA in 1X PBS. Cells were gently scraped with a cell scraper to form small aggregates (10–20 cells). Aggregates were resuspended in hESC media in the absence of bFGF and in the presence of thiazovivin (2 μM). The EBs were cultured in this media for 5 days and the EBs allowed to settle in a falcon tube. The hESC media was aspirated off and resuspended in DMEM containing 10% FBS and replated on Matrigel coated plates and EB cells were allowed to attach. Media was changed every 3 days and the culture was maintained for 14 days. Random differentiation was detected by simultaneous 3-color staining using three fluorochrome-conjugated antibodies provided with the 3 Germ Layer Immunocytochemistry Kit (Invitrogen; Catalog # A25538). Ectoderm derivatives were detected with TUJ1-rabbit (AF647), mesoderm differentiated cells with SMA-mouse (AF555), and endoderm derivatives with AFP-mouse IgG1 (AF488). The nuclei were counterstained with DAPI (blue).
Alexeeva V., Aydin I.T., Schaniel C., Stranahan A.W., D’Souza S.L, & Bieker J.J. (2020). A human H1-HBB11-GFP reporter embryonic stem cell line (WAe001-A-2) generated using TALEN-based genome editing. Stem cell research, 45, 101837.
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9

Differentiating iPSCs into Germ Layers

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iPSC lines were dissociated into single cells with TrypLE™ Express (Life Technologies), and 10,000 cells/well were plated into low-cell-adhesion 96-well culture plates with V-bottomed conical wells (Sumitomo Bakelite) to form uniform EBs cultured in: DMEM media, 20% FBS, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, P/S. After 20 days, EBs were fixed with 4%PFA and stained for three markers: smooth muscle actin (mesoderm), beta-III Tubulin (ectoderm), alpha-fetoprotein (endoderm) by Molecular Probes® 3-Germ Layer Immunocytochemistry Kit (Cat. No. A25538).
Mazza M.C., Beilina A., Roosen D.A., Hauser D, & Cookson M.R. (2021). Generation of iPSC line from a Parkinson patient with PARK7 mutation and CRISPR-edited Gibco human episomal iPSC line to mimic PARK7 mutation. Stem cell research, 55, 102506.
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10

Differentiating iPSCs into Germ Layers

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iPSC lines were dissociated into single cells with TrypLE™ Express (Life Technologies), and 10,000 cells/well were plated into low-cell-adhesion 96-well culture plates with V-bottomed conical wells (Sumitomo Bakelite) to form uniform EBs cultured in: DMEM media, 20% FBS, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, P/S. After 20 days, EBs were fixed with 4%PFA and stained for three markers: smooth muscle actin (mesoderm), beta-III Tubulin (ectoderm), alpha-fetoprotein (endoderm) by Molecular Probes® 3-Germ Layer Immunocytochemistry Kit (Cat. No. A25538).
Mazza M.C., Beilina A., Roosen D.A., Hauser D, & Cookson M.R. (2021). Generation of iPSC line from a Parkinson patient with PARK7 mutation and CRISPR-edited Gibco human episomal iPSC line to mimic PARK7 mutation. Stem cell research, 55, 102506.
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