Holotransferrin

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Holotransferrin is a protein that plays a role in iron transport and metabolism. It binds and carries iron in the bloodstream, facilitating its delivery to cells. Holotransferrin is a key component in various cellular processes and laboratory applications.

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6 protocols using holotransferrin

Isolation and Culture of Retinal Ganglion Cells

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P3/P4 retinas were freshly dissected and RGCs were isolated and purified (>99%). For details see Steinmetz et al. (2006) (link); Claudepierre et al., 2008 (link)). Briefly, cells were cultured in Neurobasal medium (Gibco/Invitrogen) supplemented with (all from Sigma, except where indicated) pyruvate (1 mM), glutamine (2 mM; Gibco/Invitrogen), N-acetyl-l-cysteine (60 μg ml⁻¹), putrescine (16 μg ml⁻¹), selenite (40 ng ml⁻¹), bovine serum albumin (100 μg ml⁻¹; fraction V, crystalline grade), streptomycin (100 μg ml⁻¹), penicillin (100 U ml⁻¹), triiodothyronine (40 ng ml⁻¹), holotransferrin (100 μg ml⁻¹), insulin (5 μg ml⁻¹) and progesterone (62 ng ml⁻¹), B27 (1:50, Gibco/Invitrogen), brain-derived neurotrophic factor (BDNF; 25 ng ml⁻¹; PeproTech, London, UK), ciliary neurotrophic factor (CNTF; 10 ng ml⁻¹; PeproTech) and forskolin (10 μm; Sigma). After isolation, RGCs were either treated for RNA extraction or fixed with PFA4% 15' at RT and processed for immunohistochemistry. Stainings were performed and cells were visualized as described above.

Savier E., Eglen S.J., Bathélémy A., Perraut M., Pfrieger F.W., Lemke G, & Reber M. (2017). A molecular mechanism for the topographic alignment of convergent neural maps. eLife, 6, e20470.

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Cell Culture Conditions for Cell Lines

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SNU308, SNU478, and SNU1196 were provided by Yonsei University. SNU308, SNU478, and SNU1196 were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher), 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin (Gibco) at 37 °C in 5% CO2. H69 cells were generously provided by Prof. Yangmi Kim and Prof. Seon Mee Park at Chungbuk National University College of Medicine. H69 cells were maintained in enriched Dulbecco's minimum essential medium (DMEM) (Hyclone) containing 10% fetal bovine serum (FBS) (Gibco, Invitrogen), 0.025 mg ml⁻¹ adenine (Sigma, St. Louis, Mo, USA), 0.005 mg ml⁻¹ insulin (Gibco Invitrogen), 0.002 mg ml⁻¹ epinephrine (sigma), 13.6 ng ml⁻¹ T3T triiodo_L_thyronine (T3) (sigma), 0.0083 mg ml⁻¹ holo‐transferrin (Gibco, Invitrogen). Hydrocortisone (Sigma) (620 ng ml⁻¹) and 10 mg ml⁻¹ epidermal growth factor (EGF; CytoLab Ltd., Rehovot, Israel) at 37 °C in 5% CO₂. All cell lines were tested and free from mycoplasma contamination (Universal Mycoplasma Detection Kit, ATCC).

Jeong M.H., Son T., Tae Y.K., Park C.H., Lee H.S., Chung M.J., Park J.Y., Castro C.M., Weissleder R., Jo J.H., Bang S, & Im H. (2023). Plasmon‐Enhanced Single Extracellular Vesicle Analysis for Cholangiocarcinoma Diagnosis. Advanced Science, 10(8), 2205148.

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Retinal Ganglion Cell Isolation and Purification

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P1/P2 retinas were freshly dissected and RGCs were isolated and purified (>99%). For details, see Steinmetz et al., 2006 (link) and Claudepierre et al., 2008 (link). Briefly, cells were harvested in neurobasal medium (Gibco/Invitrogen) supplemented with (all from Sigma, except where indicated) pyruvate (1 mM), glutamine (2 mM; Gibco/Invitrogen), N-acetyl-l-cysteine (60 mg/ml), putrescine (16 mg/ml), selenite (40 ng/ml), bovine serum albumin (100 mg/ml; fraction V, crystalline grade), streptomycin (100 mg/ml), penicillin (100 U/ml), triiodothyronine (40 ng/ml), holotransferrin (100 mg/ml), insulin (5 mg/ml) and progesterone (62 ng/ml), B27 (1:50, Gibco/Invitrogen), brain-derived neurotrophic factor (BDNF; 25 ng/ml; PeproTech, London, UK), ciliary neurotrophic factor (CNTF; 10 ng/ml; PeproTech) and forskolin (10 mM; Sigma). After isolation, RGCs were treated for RNA extraction.

Savier E.L., Dunbar J., Cheung K, & Reber M. (2020). New insights on the modeling of the molecular mechanisms underlying neural maps alignment in the midbrain. eLife, 9, e59754.

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Culturing H69 Cholangiocytes and Caco-2 Cells

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Human H69 cholangiocytes [17 –19 (link)] and Caco-2 [20 (link),21 ] cells were obtained from American Type Culture Collection (Manassas, VA, USA). The H69 cholangiocyte cell line is a SV40-transformed human bile-duct epithelial cell line originally established from a normal liver transplantation [18 (link)]. The Caco-2 cell line (designation HTB-37) is a heterogeneous human epithelial colorectal adenocarcinoma cell line [20 (link)]. Caco-2 cells were maintained in T75cm² vented monolayer flasks (Corning) with regular splitting using 0.25% trypsin (Life Technologies) every 2–5 days in complete media containing DMEM (Sigma), 10% fetal calf serum (FCS), 1 × l-Glutamine, 1 × non-essential amino acid (Gibco) and 1 × Penicillin–Streptomycin (Gibco) at 37 °C and 5% CO₂. H69 cells (SV40 immortalized cholangiocytes) were grown under similar conditions with growth factor supplemented specialist complete media [18 (link)] (DMEM/F12 with high glucose, 10% FBS, 1 × antibiotic/antimycotic, 25 µg/ml adenine, 5 µg/ml insulin, 1 µg/ml epinephrine, 8.3 µg/ml holo-transferrin, 0.62 µg/ml, hydrocortisone, 13.6 ng/ml T3 and 10 ng/ml EGF — Life Technologies).

Chaiyadet S., Smout M., Laha T., Sripa B., Loukas A, & Sotillo J. (2016). Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells. Parasitology international, 66(4), 494-502.

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Embryonic Stem Cell Differentiation

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ES cells were cultured in maintenance medium on irradiated mouse embryonic fibroblasts (MEF) at 37 ºC in 5% CO₂. The maintenance medium was composed of: Knock-Out Dulbecco’s Minimum Essential Medium (DMEM) (Life Technologies, Grand Island, NY), 15% ES cells-qualified fetal bovine serum (ES-FBS) (Gemini Bio-Products, West Sacramento, CA), 1% non-essential amino acids (NEAA) (Life Technologies), 1% penicillin/streptomycin (P/S) (Life Technologies), 2 mM Glutamax (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO) and 500 U/ml leukemia inhibitory factor (Millipore, Temecula, CA).
To initiate differentiation (day 0), MEFs were first depleted by plating dissociated ES cells on a tissue culture flask for 30–60 min. Single ES cells were then cultured in differentiation medium at 500,000 cells per 10 ml in non-adherent Petri dishes on an orbital shaker (80 rpm) at 37 ºC in 5% CO₂ to form embryoid bodies (EBs). The differentiation was composed of: Iscove’s Modified Dulbecco’s Medium (IMDM) (Life Technologies), 15% ES-FBS, 1% NEAA, 1% P/S, 2 mM Glutamax, 450 μM monothiolglyerol (Sigma), 200 μg/ml holo-transferrin (Life Technologies) and 50 μg/ml ascorbic acid (Sigma). To induce Mesp1 expression, doxycycline (500 ng/ml, Sigma) was added into the medium on day 2.25 and removed on day 3.25.

Chan S.S., Chan H.H, & Kyba M. (2016). Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq. Biochemical and biophysical research communications, 474(3), 469-475.

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In Vitro Erythroid Differentiation Assay

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In vitro erythroid differentiation analysis was performed according to the previously reported method.^{16 (link)} In brief, fetal liver cells were collected from fetuses at 14.5 gestational days for WT and R702C+/- mice. Ter119-negative cells were then purified using Ter119-conjugated magnetic beads and LD columns according to the instructions from the manufacturer (Miltenyi Biotec, Auburn, CA). Purified cells were seeded and cultured with IMDM (Life Technologies, Grand Island, NY) containing 15% fetal bovine serum, 1% BSA (Sigma, St Louis, MO), 200 µg/mL holo-transferrin (Life Technologies), 10 µg/mL recombinant human insulin (Life Technologies), 10^–4 M 2–mercaptoethanol (Life Technologies), and 2 U/mL recombinant mouse EPO (R&D Systems, Mckinley, MN) in fibronectin-coated wells (BD Discovery Labware, Bedford, MA). On the second day, EPO was removed. Flow cytometric analysis was carried out on day 0, day 1 and day 2, using PE-conjugated rat anti-mouse Ter119 antibody and FITC-conjugated rat anti-mouse CD71 (BioLegend) antibody.

Kanematsu T., Suzuki N., Tamura S., Suzuki A., Ishikawa Y., Katsumi A., Kiyoi H., Saito H., Kunishima S., Kojima T, & Matsushita T. (2021). Myh9 R702C is associated with erythroid abnormality with splenomegaly in mice. Nagoya Journal of Medical Science, 83(1), 75-86.

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