Fetal SMGs were cultured on polycarbonate membranes (13 mm, 0.1-μm pore size) (Whatman) or mesenchyme-free SMG epithelia were obtained as previously described [40 (link), 41 (link)]. The culture medium used for the isolated epithelium was DMEM/F12 (ThermoFisher Scientific) supplemented with streptomycin (100 μg/mL), vitamin C (150 μg/mL), transferrin (50 μg/mL) and FGF10 (400 ng/ml: R&D Systems). Glands were photographed at 1, 24 and 48 hr, and the morphogenic index (AU × 103) was measured as described previously [42 (link)]. Each experiment was repeated at least three times. Explants were either lysed for RNA or fixed for immunostaining. The endbud number was counted at the beginning of the experiment (T1) and at the end of the assay at 48-hours (T48) and expressed as a ratio (T48/T1) and normalized to the wildtype control. All experiments were repeated at least three times.
Transferrin
Manufactured by R&D Systems
Sourced in China
Transferrin is a protein that binds and transports iron in the body. It is a key component in various cell culture media and is used in the study of iron metabolism and related biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Human ab serum, by Merck Group (2 mentions)
Fgf10, by R&D Systems (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Polycarbonate membrane, by Cytiva (1 mentions)
Streptomycin, by R&D Systems (1 mentions)
Whatman, by Cytiva (1 mentions)
Fibrinogen, by Aviva Systems Biology (1 mentions)
Bovine serum albumin (bsa), by R&D Systems (1 mentions)
Vascular endothelial growth factor a, by R&D Systems (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Rpmi 1640 medium, by GE Healthcare (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
Iscove s medium, by Harvard Bioscience (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
6 protocols using transferrin
1
Fetal Submandibular Gland Morphogenesis Assay
2
Biomarker Screening via ELISA
3
Erythroid Differentiation of HSPCs
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Erythroid differentiation was performed using HSPCs that emerged from the adherent monolayer on day 8 of differentiation. The floating HSPCs were collected and transferred to a T25 flask for erythroid differentiation. We used the three-stage erythroid liquid culture following the previously published protocol [34 (link)]. Briefly, the HSPCs were cultured in an erythroid differentiation medium: IMDM containing 3% (v/v) human AB serum (Sigma-Aldrich), 2% (v/v) defined FBS (Hyclone), and 200 μg/mL transferrin (R&D Systems). On days 0–8 of differentiation (first stage), the medium was supplemented with 10 ng/mL stem cell factor (SCF), 1 ng/mL IL-3, and 3 U/mL EPO. On days 8–11 of differentiation (second stage), the medium was supplemented with 10 ng/mL SCF, 3 U/mL erythropoietin (EPO). In the last stage, the differentiated cells were cultured in the medium supplemented with 3 U/mL EPO and additional transferrin to a final concentration of 500 μg/mL until day 19.
4
Directed Differentiation of iSMs and iEndos
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Induced smooth muscle cells (iSMs) and induced endothelial cells (iEndos) were generated following the same protocol for iCMs, except after day 15, the medium was switched into smooth muscle growth medium containing 50% IMDM and 50% F-12, supplemented with insulin (7 μg/ml), transferrin (15 μg/ml), 450 μM MTG, BSA (5 mg/ml), and platelet-derived growth factor–BB (10 ng/ml; R&D Systems). For iEndo, EGM-2 BulletKit (Lonza) with vascular endothelial growth factor A (10 ng/ml; R&D Systems) was used.
5
Breast Cancer Cell Culture Protocol
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BPA (lot no., 162k0715; purity, 95%) and collagenase II were purchased from Sigma-Aldrich; Epidermal growth factor and cholera toxin were purchased from Merck KGaA (Darmstadt, Germany). Insulin and transferrin were purchased from R&D Systems China Co., Ltd. (Shanghai, China). The RPMI-1640 medium (without phenol red, with glutamine) was from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). RPMI-1640 medium (with glutamine) was supplied by Hyclone; GE Healthcare Sciences (Logan, UT, USA). Fetal bovine serum (FBS) was sourced from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Mouse anti-pan cytokeratin (cat. no. BM0034), rabbit anti-androgen receptor (cat. no. BA0004), rabbit anti-ERα (cat. no. BA0345), rabbit anti-ERβ (cat. no. BA2210), StreptAvidin Biotin peroxidase Complex (SABC; SA1022) kit, and 3′3 diaminobenzidine (DAB; cat. no. AR1022) kit were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Dimethyl sulphoxide (DMSO) was sourced from Shanghai Shisheng Cell Biology Technology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Annexin V-fluorescein isothiocyante (FITC), propidium iodide (PI) and 5X binding buffer were purchased from Merck & Co., Inc. (Whitehouse Station, NJ, USA).
6
Erythroid Differentiation of CD34+ Cells
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The CD34+ cells were cultured using the 3-stage erythroid culture system. During the first 8 days the cells were maintained in Basic medium which was Iscove’s medium (Biochrom) containing 3% (v/v) human AB serum (Sigma-Aldrich), 10% fetal calf serum (Hyclone, Fisher Scientific, Ltd), 10 μg/ml insulin (Sigma-Aldrich), 3 U/ml heparin (Sigma-Aldrich), 3 U/ml EPO (Roche), 200 μg/ml transferrin (R&D Systems) and 1 U/ml penicillin/streptomycin (Sigma-Aldrich) supplemented with 10 ng/ml SCF (R&D Systems) and 1 ng/ml IL-3 (R&D Systems) (primary medium). IL-3 and SCF were withdrawn from the medium on day 8 (secondary medium) and 11 (tertiary medium), respectively. In addition, extra transferrin was added to the medium to the final concentration of 500 μg/ml from day 11 onward. The cells were counted and medium was added every other day. The cultured cells were maintained at 37 °C, 5% CO2 throughout the culture period. At indicated time points, aliquots of cells were collected for morphological analysis using cytospin and Leishman staining. Two sample equal variance t-test was carried out to determine the statistic significances of cell numbers and cell types.
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