1 thioglycerol

Manufactured by Merck Group

Sourced in United States, Japan, Germany, United Kingdom

1-thioglycerol is a colorless, viscous liquid chemical compound. It is commonly used as a laboratory reagent in various applications, including the synthesis of organic compounds and the study of biochemical processes. The core function of 1-thioglycerol is to serve as a versatile building block and intermediate in chemical reactions.

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98 protocols using 1 thioglycerol

Whole Mount Tissue Clearing and Staining

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Whole mount aortic arch, mediastinal adipose tissue, and thymus were fixed in Cytofix (BD Biosciences) 1:3 diluted in PBS overnight. The tissue block was washed in PBS for a day and blocked with 1% mouse serum (Milipore Sigma), 1% BSA (Milipore SIGMA), 0.3% TritonX-100 in PBS for 24h at room temperature while agitating. Subsequently, primary fluorescently conjugated antibodies were added 1:100 for 72h at 37°C in the dark while agitating. Hoechst (Thermo Scientific) was added at a concentration of 1:1000 for the final 2h. After staining, the whole mount tissue block was washed in PBS with 0.2% Triton X-100 and 0.5% 1-Thioglycerol (Milipore Sigma) overnight at room temperature. The buffer was replaced twice. C_e3D clearing solution (6 (link)) was freshly prepared as follows:
1ml contained 400μl (40%) N-methylacetamide, 1μl of Triton X-100 (0.1%), 5μl 1-Thioglycerol (0.5%) (all obtained from Milipore Sigma). For optimal solvation of all reagents, the buffer was shaken at 37°C for several hours. The whole mount tissue was removed from the washing buffer, carefully dried, and transferred into the clearing solution overnight at room temperature in the dark. The following day, the cleared whole mount tissue was mounted between two 1.5 borosilicate glass coverslips in clearing medium.

Winkels H., Ghosheh Y., Kobiyama K., Kiosses W.B., Orecchioni M., Ehinger E., Suryawanshi V., Herrera-De La Mata S., Marchovecchio P., Riffelmacher T., Thiault N., Kronenberg M., Wolf D., Seumois G., Vijayanand P, & Ley K. (2021). Thymus-derived CD4+CD8+ cells reside in mediastinal adipose tissue and the aortic arch. Journal of immunology (Baltimore, Md. : 1950), 207(11), 2720-2732.

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Differentiation of Endothelial Cells from Human Pluripotent Stem Cells

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Human endothelial cells were generated as previously described^{7 (link)}. Maintenance and formation of EBs was performed as described in the paragraph cardiac differentiation. For induction of differentiation of mesodermal progenitor cells, EBs were cultivated in Pluronic F-127 (Sigma-Aldrich, P2443) -coated flasks in StemPro^®-34 (Gibco 10639-011) supplemented with 4 mg/ml PVA; 400 µM 1-thioglycerol, Sigma-Aldrich M6145; 2 mM L-glutamin, Gibco 25030; 5 mg/L transferrin, Sigma-Aldrich T8158; 5 µg/L selenium, Sigma-Aldrich S5261; 0.5% Penicillin/Streptomycin, Gibco 15140; 10 µM Y-27632, biorbyt orb60104 and 10 ng/ml BMP-4, R&D Systems 314-BP; 6 ng/ml Activin-A, R&D systems 338-AC; 5 ng/ml basic FGF, R&D systems 233-FB for 3 days at 37 °C, 90% humidity, 5% CO₂, 5% O₂ with daily medium change. To differentiate endothelial cells, EBs were then cultured on Geltrex^® in StemPro^®-34, containing 400 µM 1-thioglycerol, Sigma-Aldrich M6145; 2 mM L-glutamin, Gibco 25030; 1 mM magnesium ascorbyl phosphate; 5 mg/L transferrin, Sigma-Aldrich T8158; 5 µg/L selenium, Sigma-Aldrich S52610.5% Penicillin/Streptomycin, Gibco 15140; 1 µM Y-27632, biorbyt orb60104; 100 ng/ml VEGF, R&D Systems 293-VE and 10 ng/ml bFGF, R&D Systems 233-FB. For 3 days, EBs were cultured in a hypoxic environment (5% CO₂, 5% O₂), followed by 9 days under normoxic conditions. Medium was changed every other day.

Pecha S., Yorgan K., Röhl M., Geertz B., Hansen A., Weinberger F., Sehner S., Ehmke H., Reichenspurner H., Eschenhagen T, & Schwoerer A.P. (2019). Human iPS cell-derived engineered heart tissue does not affect ventricular arrhythmias in a guinea pig cryo-injury model. Scientific Reports, 9, 9831.

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Differentiation of iPSCs into Hematopoietic Progenitors

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To differentiate iPSCs into hematopoietic progenitors, we adjusted the protocol from Liu et al. [44 (link)]: EBs were generated from iPSCs by self-detachment from microcontact-printed vitronectin spots generated with PDMS stamps [45 (link)]. EBs were carefully resuspended in serum free medium containing 50% IMDM, 50% Ham’s F12, 0.5% BSA, 1% chemically defined lipid concentrate, 2 mM GlutaMAX (all Thermo Fisher Scientific), 400 μM 1-thioglycerol, 50 μg/mL L-ascorbic acid, and 6 μg/mL holo transferrin (all Sigma Aldrich, St. Louis, MO, USA) supplemented with 10 ng/mL FGF-2 (Peprotech, Hamburg, Germany), 10 ng/mL BMP-4 (Miltenyi Biotec), and 10 μM Y-27632 (Abcam, Cambridge, Great Britain). EBs were seeded on 0.1% gelatin coated 6-well plates. From day 2 to 16, EBs were cultured in serum-free medium supplemented with 10 ng/mL FGF 2, 10 ng/mL BMP-4, 50 ng/mL SCF, 10 ng/mL VEGF-A (all Peprotech), and 10 U/mL penicillin/streptomycin (Thermo Fisher Scientific). The non-adherent iHPCs were harvested at day 16 and separated with a 40 μm cell strainer. Immunophenotype was tested with flow cytometry and stem cell potential with colony forming unit assays as described in Additional file 1.

Cypris O., Franzen J., Frobel J., Glück P., Kuo C.C., Schmitz S., Nüchtern S., Zenke M, & Wagner W. (2022). Hematopoietic differentiation persists in human iPSCs defective in de novo DNA methylation. BMC Biology, 20, 141.

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Directed Differentiation of cjiPSCs

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cjiPSCs were differentiated in vitro using the EB formation assay according to a previously published protocol (Stauske et al, 2020 (link)). After treatment with collagenase type IV, cell clumps derived from single colonies were cultured as a suspension in UPPS medium for 24 h, to allow complete aggregation. The UPPS medium was then replaced with differentiation medium (IMDM [12440053; Thermo Fisher Scientific], 20% FBS [16000044; Gibco], 1x MEM NEAA [11140-035; Gibco], and 450 μM 1-thioglycerol [M1753; Sigma-Aldrich]). After 8 d, the EBs were transferred to a cell culture plate with coverslips coated with 0.1% gelatine (ø 25 mm; Thermo Fisher Scientific). After 25 d from the start of the protocol, coverslips containing embryoid body outgrowths were fixed and immunostained.

Kurlovich J., Rodriguez Polo I., Dovgusha O., Tereshchenko Y., Cruz C.R., Behr R, & Günesdogan U. (2024). Generation of marmoset primordial germ cell–like cells under chemically defined conditions. Life Science Alliance, 7(6), e202302371.

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Maintenance of Mouse Embryonic Stem Cells

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Mouse embryonic stem cell line E14TG2a (mES cells) was cultured with the N2B27 base medium supplemented with 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.15 mM 1-thioglycerol (Sigma), 100 U/ml of penicillin–streptomycin (Invitrogen), 25 μg/ml of BSA (Sigma), 1 μM MEK inhibitor PD0325901 (Selleck Chemicals), 3 μM GSK3β inhibitor CHIR99021 (Selleck Chemicals), 2% KOSR (Thermo Fisher), and 1000 U/ml of ESGRO leukemia inhibitory factor LIF (Millipore) on plates coated with 0.2% gelatin.

Xiao Z., Liu S., Li Z., Cui J., Wang H., Wang Z., Ren Q., Xia L., Wang Z, & Li Y. (2022). The Maternal Microbiome Programs the m6A Epitranscriptome of the Mouse Fetal Brain and Intestine. Frontiers in Cell and Developmental Biology, 10, 882994.

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Immortalized Megakaryocyte Cell Culture

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ImMKCLs were cultured and expanded via Doxycycline dependent expression of C-MYC, BMI-1 and BCL-XL^{34 (link)}. ImMKCLs were maintained in a humidified incubator at 37°C and 5% CO2 and in IMDM (Sigma-Aldrich) medium supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich), L-glutamine (Gibco), Insulin-transferrin-selenium (Gibco), 50 mg/mL Ascorbic acid (A4544; Sigma-Aldrich), and 450 mM 1-Thioglycerol (Sigma-Aldrich), 50 ng/mL carrier-free recombinant human stem cell factor (SCF; R&D Systems), 50 ng/mL TPO (R&D Systems), and 5 mg/mL Doxycycline (Clontech).

Lee D.H., Yao C., Bhan A., Schlaeger T., Keefe J., Rodriguez B.A., Hwang S.J., Chen M.H., Levy D, & Johnson A.D. (2020). Integrative Genomic Analysis Reveals Four Protein Biomarkers for Platelet Traits. Circulation research, 127(9), 1182-1194.

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Cardiomyogenic Differentiation of hiPSCs

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Human-induced pluripotent stem cells (hiPSCs; 253G1; Riken, Ibaraki, Japan) were used in this study. Undifferentiated hiPSCs were cultured and maintained in primate embryonic stem cell medium (ReproCELL, Kanagawa, Japan) with 5 ng/mL basic fibroblast growth factor (bFGF; ReproCELL) on mitomycin C-treated mouse embryonic fibroblast cells (ReproCELL). Cardiomyogenic differentiation was induced as previously described with specific modifications (Matsuura et al., 2012 (link)). Briefly, cardiac differentiation was induced in StemPro 34 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 2 mM L-glutamine (Thermo Fisher Scientific), 50 μg/mL ascorbic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 400 μM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO, USA). The medium was also supplemented with several human recombinant proteins, including bone morphologic protein 4, activin A, bFGF (R&D Systems, Minneapolis, MN, USA), and VEGF (FUJIFILM Wako Pure Chemical Corporation), and small molecules, including IWR-1 and IWP-2 (Sigma-Aldrich). hiPSCs were dissociated using Accumax (Nacalai Tesque, Kyoto, Japan) and transferred to a bioreactor (ABLE Corporation & Biott, Tokyo, Japan). hiPSC-CMs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich).

Nakazato T., Kawamura T., Uemura T., Liu L., Li J., Sasai M., Harada A., Ito E., Iseoka H., Toda K., Sawa Y, & Miyagawa S. (2022). Engineered three-dimensional cardiac tissues maturing in a rotating wall vessel bioreactor remodel diseased hearts in rats with myocardial infarction. Stem Cell Reports, 17(5), 1170-1182.

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Erythroblast Differentiation from Human iPSCs

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Human iPSCs (passages 25–35) were differentiated into erythroblasts according to a previous study 30, with a minor modification. Briefly, iPSCs were maintained on feeder free condition in E8 medium. At 70% confluency, the cells were detached by incubating with 1 mg/ml Collagenase IV (Life Technologies) treatment for 15 minutes, scraped by cell scraper (Corning, Corning, NY, http://www.corning.com), washed with DMEM/F12 basal medium (Hyclone) for 2 times, and resuspended in erythroid differentiation medium (alpha‐minimum essential medium (MEM) without nucleoside [Hyclone], 10% defined fetal bovine serum [Hyclone], 100 µM 1‐thioglycerol [Sigma‐Aldrich], 50 µg/ml ascorbic acid [Sigma‐Aldrich], 2 mM Glutamax, 1% penicillin/streptomycin). The cell suspension was seeded onto 3–5 days post‐confluent OP9 cells (ATCC, Manassas, VA, http://www.atcc.org), at 1–1.5 × 10⁶ cells per 10‐cm dish in 20 ml erythroid differentiation medium. On the next day, total medium was replaced and at day 4 and 6, half of medium was changed. After 8 days of coculture on OP9 cells, the cells were harvested for colony forming cells (CFC) assay.

Phanthong P., Borwornpinyo S., Kitiyanant N., Jearawiriyapaisarn N., Nuntakarn L., Saetan J., Nualkaew T., Sa‐ngiamsuntorn K., Anurathapan U., Dinnyes A., Kitiyanant Y, & Hongeng S. (2017). Enhancement of β‐Globin Gene Expression in Thalassemic IVS2‐654 Induced Pluripotent Stem Cell‐Derived Erythroid Cells by Modified U7 snRNA. Stem Cells Translational Medicine, 6(4), 1059-1069.

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Erythroblast Induction from CD34+ Progenitors

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To generate erythroid cells, sorted CD34⁺ progenitors were plated in 0.1% gelatin-coated plates (1.0×10⁶/well in a 6-well plate) and cultured for 4 days in erythroblast induction medium, which consisted of 49% IMDM (Life Technologies, 12440), 49% Ham’s F12 (Life Technologies, 11765), 1% ITSX, 1% lipid (Life Technologies, 11905-031), 5 mg/100 mL I-ascorbic acid (Sigma, A8960), 500 mg/100 mL BSA (Sigma, A7030), and 200uM 1-thioglycerol (Sigma, M6145), supplemented with 100 ng/mL SCF (PeproTech, 300–07), 10 ng/mL interleukin-3 (PeproTech, 200–03), 5 U/mL EPO (R&D Systems, 287-TC), 40 ng/mL insulin-like growth factor-1(Sigma I-3769), 1 μM Dexamethsone (Sigma, D2915), and 50 ng/mL Flt3-L (PeproTech, 300–19) (Figure 1A). Unlike adherent ECs, induced erythroblasts detached on generation and were collected as nonadherent cell fraction at day 4 for characterization.

Zhang L., Jambusaria A., Hong Z., Marsboom G., Toth P.T., Herbert B.S., Malik A.B, & Rehman J. (2017). SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34+ Progenitor Cells. Circulation, 135(25), 2505-2523.

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Culturing GIST-T1 and HMC 1.2 Cell Lines

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GIST-T1 cell lines were obtained from T. Taguchi (Kochi Medical School, Nankoku, Japan), and the human mast cell line HMC 1.2 were obtained from I. Pass, Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA. Both the cell lines were cultured following previous methods.^{10 (link)} The GIST-T1 cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS, 1% penicillin/streptomycin (Sigma), and 2 mmol/L glutamine (Sigma). The human mast cell line HMC 1.2 were cultured in Iscove's modified Dulbecco's Medium (Gibco) with 10% FBS, 1% penicillin/streptomycin, and 1.2 mmol/L 1-Thioglycerol (Sigma).^{10 (link)}

Vijayakumar S., Nasr S.H., Davis J.E., Wang E., Zuidema J.M., Lu Y.S., Lo Y.H., Sicklick J.K., Sailor M.J, & Ray P. (2022). Anti-KIT DNA Aptamer-conjugated Porous Silicon Nanoparticles for the Targeted Detection of Gastrointestinal Stromal Tumors. Nanoscale, 14(47), 17700-17713.

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