Human apo transferrin

Minced mammary tissue was digested (1.5 mg/ml collagenase A (Roche, San Francisco, CA; 75 μg/ml DNase I, Roche; 1 mg/ml hyaluronidase, MP Biomedicals, Santa Ana, CA) in growth media (10 % fetal bovine serum [FBS], DMEM/F-12, penicillin G/streptomycin sulfate/amphotericin B) at 37 °C for 3 h. Organoids (40–100 μm diameter) were plated in primary porcine mammary epithelial media (modified from MEGM [21 ] as a 1:1 mix of MCDB170 (US Biological, Salem, MA) and DMEM/F-12 (CellGro, Manassas, VA) with penicillin G/streptomycin sulfate/amphotericin B, 0.5 % FBS, bovine insulin (7.5 μg/mL, Sigma-Aldrich), human EGF (5 ng/mL, Millipore, Billerica, MA), hydrocortisone (0.25 μg/mL, Sigma-Aldrich), human apo-transferrin (2.5 μg/mL, Sigma-Aldrich), ethanolamine (0.1 mM, Sigma-Aldrich), o-phosphoethanolamine (0.1 mM Sigma-Aldrich), bovine pituitary extract (35 μg/mL, Gemini Bio-Products, West Sacramento, CA), and lipid-rich bovine serum albumin (0.1 %, Gemini Bio-Products).

Differentiation started with SGBS cells at confluence (day 0) in the medium containing 10% FBS. Preadipocytes were washed with PBS, and then changed to the primary differentiation serum-free medium (differentiation A media), for 4 days, containing: biotin 33 μM, pantothenic acid 17 μM, penicillin/streptomycin 100 U/mL, rosiglitazone 2 μM (Cayman Chemicals, Ann Arbor, MI, USA), human apotransferrin 10 μg/mL (Sigma-Aldrich, Saint Louis, MO, USA), human insulin 20 nM (Sigma-Aldrich, Saint Louis, MO, USA), dexamethasone 25 nM (Sigma-Aldrich, Saint Louis, MO, USA), 3-isobutyl-1-methylxanthines 500 μM (Sigma-Aldrich, Saint Louis, MO, USA), cortisol 100 nM (Sigma-Aldrich, Saint Louis, MO, USA) and triiodothyronine 200 pM (Sigma-Aldrich). After the fourth day, the medium was changed. rosiglitazone, 3-isobutyl-1-methylxanthines and dexamethasone were removed during the remaining 10 days of differentiation (differentiation B media). B media was replaced every four days. Cells were considered fully mature 28 days post-differentiation when clearly visible lipid droplets formed.

Serum survival assays were performed as described previously with some modifications [29–31 (link)]. ExPEC strain RS218 was cultured in LB medium to the logarithmic growth phase. Fresh bacteria were washed twice with M9 medium. Then, 1 × 10⁷ CFUs of bacteria were incubated with 20% transferrin-deficient HIHS, 20% transferrin-deficient HIHS supplemented with 200 μg/mL human holo-transferrin (Sigma, Catalog No. T0665) or 200 μg/mL human apo-transferrin (Sigma, Catalog No. T2036), or 20% control HIHS in M9 medium at 37°C, respectively. After 3 h of incubation, bacteria were counted by plate counting. Data were calculated as the ratio of the number of bacteria recovered from serum to the original number of bacteria. To identify the effect of holo-transferrin and apo-transferrin in HIHS on the viability of ExPEC, the growth ability of ExPEC RS218 in M9 medium and M9 medium supplemented with holo-transferrin or apo-transferrin was also tested as described above. These experiments were repeated six times independently.

3T3-CD40L⁺ were harvested, irradiated with 30 Gy and seeded in B cell medium (RPMI 1640 (Gibco, through Thermo Fisher) without phenol red containing 5% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 50 µM β-mercaptoethanol and 20 µg/mL human apo-transferrin (Sigma Aldrich; depleted for human IgG with protein G sepharose (GE Healthcare, 3871 MV Hoevelaken, The Netherlands)) on 96-well flat-bottom plates (Nunc through Thermo Fisher) to allow adherence overnight. 3T3-CD40L⁺ were seeded at 10,000 cells per well. The next day, CD19⁺ B cells were thawed from cryo-storage and washed with B cell medium. B cells were rested at 37 °C for 1 h before counting. Then, 5000 or 10,000 CD19⁺ B cells were co-cultured with the irradiated 3T3-CD40L⁺ fibroblasts in the presence of F(ab’)₂ fragment Goat Anti-Human IgA/G/M (5 µg/mL; Jackson Immunoresearch, Ely CB7 4EZ, UK), IL-4 (25 ng/mL; Cellgenix, 79107 Freiburg, Germany) and IL-21 (50 ng/mL; Peprotech, London W6 8LL, UK) for up to six days. The B cell suspension, 3T3-CD40L⁺ plates and stimulation mix were all heated to 37 °C before mixing the components together. After adding the B cells to the wells, the plate was centrifuged for 1 min at 400× g to force all the B cells onto the 3T3-CD40L⁺ layer.

Ferroxidase activity in triton X-100 extracts (described above) was determined as previously described [30 (link), 33 (link)]. In brief, for each assay run, fresh solutions of 250 µM human apo-transferrin (Sigma) and 1 mM FeSO₄ were prepared in N₂-purged dH₂O to mitigate ferroxidase-independent oxidation of iron. Reaction mixtures in HEPES-buffered saline (50 mM HEPES, 150 mM NaCl, pH 7.2) contained 50 µM apo-transferrin and sample (or triton X-100 supplemented TBS-based homogenisation buffer as vehicle control), then reactions initiated by adding FeSO₄ to a final concentration of 100 mM. The formation of holo-transferrin was monitored via change in absorbance at 460nm for 5 min at 25 °C.

For the cellular experiments, we used immortalized cultures of human myoblasts. These cells, called LHCNM2, were previously derived from the pectoralis major muscle of a 41-year-old male Caucasian heart-transplant donor and immortalized by introduction of human telomerase and cyclin-dependent protein kinase 4 [24 (link)]. The cells were cultured in medium containing four parts Dulbecco’s modified Eagle’s medium (DMEM; 4.5 mg/ml glucose) and one part medium 199, supplemented with 20% fetal bovine serum. For the differentiation studies, the cells were trypsinized, counted, and 50,000 cells were plated on to a 48-well plate. After 6–7 days, when the cells had become confluent, the proliferation medium was removed and replaced with DMEM (Gibco) supplemented with 10 mg/ml bovine insulin (Sigma-Aldrich, Saint-Quentin Fallavier France) and 100 mg/ml of human apo-transferrin (Sigma-Aldrich), as previously described in the literature [24 (link)].