Human transferrin

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada

Human transferrin is a protein that is responsible for the transport and regulation of iron in the human body. It is commonly used as a reagent in various laboratory applications.

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23 protocols using human transferrin

Chondrogenic Differentiation of ATDC5 Cells

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ATDC5 cells were cultured in growth medium (1:1 Dulbecco's modified Eagle's medium (DMEM): Ham's F-12 mix (Gibco)) containing 1% (vol/vol) antibioticeantimycotic (Gibco), 5% fetal bovine serum (FBS) (Gibco), 10 mg/ml human transferrin (Sigma) and 30 nM sodium selenite (Sigma). Cells were maintained in a humidified atmosphere of 5% CO 2 at 37 C. High-density micromass cultures of ATDC5 cells were grown to study chondrogenic differentiation 18 . Cells were trypsinized, washed and resuspended at 2 Â 10 7 cells/ml in a chondrogenic medium made of DMEM-F12 enriched by 1% (vol/vol) antibioticeantimycotic, 5% FBS, 5 mg/ml human transferrin and 1 Â ITS (Insulin, Transferrin, Selenite) premix (resulting in 10 mg/ml insulin, 5 mg/ml human transferrin and 30 nM sodium selenite) (Life Technologies). One droplet (10 ml) was carefully placed in the centre of each well of a 24-well plate. Cells were allowed to adhere for 2 h at 37 C, followed by addition of 500 ml chondrogenic medium. Micromasses and supernatants were collected at time points 1, 7 and 14 days for functional assays.

Wang X., Cornelis F.M.F., Lories R.J, & Monteagudo S. (2019). Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation. Osteoarthritis and cartilage, 27(11).

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Cultivation of Immortalized Human Renal Proximal Tubular Epithelial Cells

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PTEC-TERT1 cells (renal PTEC [RPTEC]/TERT1, CHT-003-0002; Evercyte, Wien, Austria) were cultured according to the manufacturer’s instructions in PTEC medium: Mix 1/1 of DMEM (#11966-025) and Ham’s F-12 nutrients (#21765-29) (Thermo Fisher Scientific, Waltham, MA, USA) containing 1% penicillin-streptomycin (GIBCO, #15140-122), 10 mM HEPES (GIBCO, #15630-056), 5.0 μg/mL human insulin (GIBCO, #41400-045), 5.0 μg/mL human transferrin (GIBCO, #41400-045), 8.65 ng/mL sodium selenite (GIBCO, #41400-045), 0.1 μM hydrocortisone (#H6909, Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL human recombinant EGF (#236-EG-200, R&D Systems, Minneapolis, MN, USA), 3.5 μg/mL ascorbic acid (Sigma-Aldrich, #A4544 powder), 25 ng/mL prostaglandin E1 (Sigma-Aldrich, #P5515), 3.2 pg/mL Triiodo-L-thyronine (Sigma-Aldrich, #T-5516), and 100 μg/mL Geneticin (GIBCO, #10131-027).

Sewing S., Gubler M., Gérard R., Avignon B., Mueller Y., Braendli-Baiocco A., Odin M, & Moisan A. (2018). GalNAc Conjugation Attenuates the Cytotoxicity of Antisense Oligonucleotide Drugs in Renal Tubular Cells. Molecular Therapy. Nucleic Acids, 14, 67-79.

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Caco-2 Cell Line Transcriptional Regulation

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Well-differentiated mature monolayers of Caco-2BBE (human intestinal epithelial) cells were maintained in 25 mM glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Life Technologies, Seoul, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, Seoul, Korea), 1% penicillin-streptomycin (Gibco, Life Technologies, Seoul, Korea), and 0.01 mg/ml human transferrin (Gibco, Life Technologies, Seoul, Korea). Cell cultures were maintained in a 37°C incubator supplemented with 5% CO₂. Luciferase reporter plasmids containing the Glut5 promoter constructs were transfected into Caco-2BBE cells with or without pcDNA expression vectors containing the Chrebp and Mlx genes. Luciferase activity was determined using the Luciferase Assay System (Promega Corporation, Fitchburg, WI) according to the manufacturer’s instructions and expressed as arbitrary units normalized to β-galactosidase activity. All experiments were performed in triplicate and repeated at least three times.

Oh A.R., Sohn S., Lee J., Park J.M., Nam K.T., Hahm K.B., Kim Y.B., Lee H.J, & Cha J.Y. (2018). ChREBP deficiency leads to diarrhea-predominant irritable bowel syndrome. Metabolism: clinical and experimental, 85, 286-297.

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Characterization of Intestinal Cell Lines

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The Caco-2 (CRL-2102) cell line was obtained from ATCC (Rockville, MD), and HT29-MTX cell line was obtained from the Publich Health England Culture Collections (Salisbury, Great Britain). Both Caco-2 and HT29-MTX cells were grown in DMEM supplemented with 10% fetal bovine serum, 10 μg/mL human transferrin (Gibco), and 1% antibiotics and antimycotics. In addition, human Intestinal Myofibroblasts (H-InMyoFib) were purchased from Lonza and cultured in SMGM™-2 BulletKit™ medium (Lonza). Cells were cultured in T-175 cm² tissue culture flasks (Corning), maintained at 37 °C, 5% CO₂ humidied atmosphere and harvested with 0.25% trypsin-EDTA (Gibco) prior to seeding. For Caco-2 and HT29-MTX, cells from passage number 38–50 were used for the experiments. For H-InmyoFib, passage 3–5 was used.

Chen Y., Lin Y., Davis K.M., Wang Q., Rnjak-Kovacina J., Li C., Isberg R.R., Kumamoto C.A., Mecsas J, & Kaplan D.L. (2015). Robust bioengineered 3D functional human intestinal epithelium. Scientific Reports, 5, 13708.

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Co-culture of Intestinal Epithelial and Myofibroblast Cells

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Human Intestinal Myofibroblasts (InMyoFibs) (Lonza, Portsmouth, NH) were plated in the basal compartment of a 24-well Transwell culture system (pore size 0.4 μm; Costar Corp.) at a density of 1 × 10⁴ cells/cm². Caco-2 (ATCC; Rockville, MD) and HT29-MTX (Public Health England Culture Collection; Salisbury, Great Britain) intestinal epithelial cell lines were then plated at a ratio of 3:1, respectively, in the apical compartment of the Transwell system at a density of 5 × 10⁴ cells/cm². The seeded epithelial cells were incubated at 37 °C for 1 h to allow for attachment before being placed into the 24-well plates (the basal compartment of the Transwell system) containing the InMyoFibs to commence experiments. The Transwell co-culture was fed every 2–3 days with DMEM/SMGM medium (1:1, DMEM: DMEM supplemented with 10% FBS (fetal bovine serum) and 10 μg/mL human transferrin (Gibco, Gaithersburg, MD), SMGM: Smooth Muscle Cell Growth Medium (Lonza; Basel, Switzerland)) for 1-, 2-, 3-, and 4-week collection time points. For the entire study, Caco-2 and HT29-MTX cells from passage numbers 45–60 were used for seedings, and for InMyoFibs, passage numbers 4–7 were used. All cells tested negative for mycoplasma contamination.

Rudolph S.E., Longo B.N., Tse M.W., Houchin M.R., Shokoufandeh M.M., Chen Y, & Kaplan D.L. (2022). Crypt-Villus Scaffold Architecture for Bioengineering Functional Human Intestinal Epithelium. ACS biomaterials science & engineering, 8(11), 4942-4955.

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Rhodamine 123 Uptake in Cell Lines

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Rhodamine 123 (Rho 123), elacridar (Ela), zosuquidar (Zos), neutral red (NR) solution, and Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose were obtained from Sigma (St. Louis, MO, USA). Reagents used in cell culture, such as nonessential amino acids (NEAA), heat-inactivated bovine serum (FBS), 0.25% trypsin/1 mM ethylenediamine tetraacetic acid (EDTA), antibiotic (10,000 U/mL penicillin, 10,000 μg/mL streptomycin), human transferrin (4 mg/mL), Hank’s balanced salt solution without calcium and magnesium (HBSS (-/-)), and phosphate-buffered saline solution (PBS), were purchased from Gibco Laboratories (Lenexa, KS). P-gp monoclonal antibody (clone UIC2) conjugated with PE was purchased from Abcam (Cambridge, United Kingdom). All the reagents used were of analytical grade or the highest grade available.

Martins E., Silva V., Lemos A., Palmeira A., Puthongking P., Sousa E., Rocha-Pereira C., Ghanem C.I., Carmo H., Remião F, & Silva R. (2019). Newly Synthesized Oxygenated Xanthones as Potential P-Glycoprotein Activators: In Vitro, Ex Vivo, and In Silico Studies. Molecules, 24(4), 707.

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Caco-2 and HT29-MTX Cell Culture Protocol

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The Caco-2 (CRL-2102) cell line was obtained
from ATCC (Rockville, MD). The HT29-MTX cell line was obtained from
the Public Health England Culture Collections (Salisbury, Great Britain).
Human Intestinal Myofibroblasts (H-InMyoFib) were purchased from Lonza.
Both Caco-2 and HT29-MTX cells were grown in DMEM supplemented with
10% fetal bovine serum, 10 μg/mL human transferrin (Gibco),
and 1% antibiotics and antimycotics. Myofibroblasts were cultured
in SMGM-2 BulletKit medium (Lonza). Polydimethylsiloxane (PDMS, Sylgard
184) was purchased from Dow Corning, and Ecoflex 00–30 was
purchased from Smooth-On. A Teflon cylinder, Teflon tubes, adaptor,
and silicone rubber tubes were purchased from McMaster-Carr.

Zhou W., Chen Y., Roh T., Lin Y., Ling S., Zhao S., Lin J.D., Khalil N., Cairns D.M., Manousiouthakis E., Tse M, & Kaplan D.L. (2017). Multifunctional Bioreactor System for Human Intestine Tissues. ACS Biomaterials Science & Engineering, 4(1), 231-239.

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Cell Line Cultivation for Experimental Assays

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HEK293T and AML12 cell lines were purchased from ATCC. HEK293T and stably expressed HEK293T cells were propagated in DMEM (Thermo Fisher) supplemented with 10% v/v fetal bovine serum (FBS, Atlanta Biologicals), 2 mM L-glutamine (Life Technologies), and 1% v/v penicillin-streptomycin (5,000 U/mL, Life Technologies). AML12 and stably expressed AML12 cells were propagated in DMEM/F-12 (Gibco) supplemented with 10% v/v FBS, 0.1% v/v gentamicin (50 mg/mL, Sigma Aldrich), 1% v/v ITS-G (100x, 1.0 mg/mL recombinant human insulin, 0.55 mg/mL human transferrin, 0.67 μg/mL sodium selenite, Gibco), and 0.04 μg/mL dexamethasone (Sigma Aldrich). AML12 and stable AML12 cells were switched to serum-free media for 24 hours before the experiments and treated with the rapalog under the same condition³⁷. All cell lines were grown at 37 °C in a 5% CO₂ humid incubator.

Lee G, & Muir T.W. (2023). Distinct phases of cellular signaling revealed by time-resolved protein synthesis. bioRxiv.

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Culturing Human Renal Epithelial HK-2 Cells

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Human renal epithelial HK-2 cells were from ATCC^® (CRL-2190^TM) and syndecan-1 deficient HK-2 cells were obtained by shRNA technology and selected based on zeocin resistance^{30 (link)}. Cells were cultured in a mixture of DMEM and Ham’s F-12 medium, supplemented with 10 ng/mL human recombinant epidermal growth factor (EGF), 36 ng/mL hydrocortisone, 5 μg/mL bovine insulin, 5 μg/mL human transferrin, 5 ng/mL sodium selenite, 2 mM glutamax (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin.

Campeiro J.D., Dam W., Monte G.G., Porta L.C., Oliveira L.C., Nering M.B., Viana G.M., Carapeto F.C., Oliveira E.B., van den Born J, & Hayashi M.A. (2019). Long term safety of targeted internalization of cell penetrating peptide crotamine into renal proximal tubular epithelial cells in vivo. Scientific Reports, 9, 3312.

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Maintaining Cortical Progenitor Cells

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E14 cortical progenitor cells (R&D systems) were seeded onto 15μg/ml Poly-L-ornithine (Sigma) and 1μg/ml laminin (Sigma) coated 6 well plates as a monolayer culture. Cell culture medium was composed of DMEM/F-12 with glutamax (Life Technologies), 1X N2 supplement composed of Insulin, Human Transferrin, Putrescine, Selenite and Progesterone (Life Technologies) and glucose (Sigma). Culture medium was supplemented with 10ng/ml of human basic fibroblast growth factor (R&D systems) and 10ng/ml of human epidermal growth factor (R&D systems) every day until cell lysate collection to maintain cortical progenitors cells in an undifferentiated state.

Mishra S., Choe Y., Pleasure S.J, & Siegenthaler J.A. (2016). Cerebrovascular defects in Foxc1 mutants correlate with aberrant WNT and VEGF-A pathways downstream of retinoic acid from the meninges. Developmental biology, 420(1), 148-165.

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