Anti cd71

On day 8, 10, 12, 14 and 16 time points, non-adherent cells were collected and washed in IMDM. Cells were stained with cell surface antibodies, anti CD235a cat# 551336 (BDbiosciences, San Jose, CA, USA) and anti CD71 cat# 555537 (BDbiosciences, San Jose, CA, USA) for 30 min at 4 °C. Next cells were fixed and permeabilized with 200 ul of Cytospin/Cytoperm (BD Biosciences San Jose, CA) followed by intracellular staining using anti AHSP cat# sc515436 (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h at room temperature, and then with Hoechst 33342 nuclear stain cat# H3570 (Thermofisher Invitrogen, Waltham, MA) for 10 min at room temperature. Cells were then analyzed by flow cytometry using BD LSRFortessa and gated with FlowJo software.

For immunophenotyping, BM cells were stained using fluorescent label-conjugated anti-CD71, anti-ter119, anti-CD45, anti-CD11b, and anti-Gr-1 antibodies (BD Biosciences, San Jose, CA). For intracellular staining, NRF2 (D1Z9C) XP® Rabbit mAb (PE Conjugate) was used, following the technique provided by the manufacturer. For measure of oxidative stress, Total ROS/Superoxide detection kit was used (Enzo Life Sciences). The stained cells were analyzed by Accuri C6 flow cytometer and CFlow plus software (BD Biosciences, San Jose, CA).

Cell-cycle status and S phase speed were analyzed using BrdU incorporation^{26 (link)}. Briefly, cells were pulsed at a final concentration of 33 μM BrdU for 30 min. Cells were immediately labeled with the LIVE/DEAD Kit (Invitrogen L23105), fixed, and permeabilized. Erythroid subsets were identified using anti-CD71 (BD Biosciences 113812) and anti-Ter119 (BD Biosciences 553673). BrdU incorporation was measured by biotin-conjugated anti-BrdU (MOBU-1, BioLegend) followed by a secondary stain with Brilliant Violet 421™ Streptavidin (BioLegend). DNA content was measured by 7AAD (BD Biosciences).

Expanded erythroblasts were labeled with anti-CD71 (FITC), 235a (PE), 36 (FITC) and 117 (PE), (BD Biosciences, East Rutherford, New Jersey, U.S.). Cells were acquired in BD FACSCalibur™ flow cytometer and viable 7-AAD (BD Biosciences) negative cells were analyzed in FlowJo V10 software (BD Biosciences).

Antibody staining was done on ice. Cells were washed once with PBS/1% FCS and afterwards incubated for 30 minutes in a total volume of 100 μl with the respective recommended amount of antibody. Following antibodies were used in our study: anti-CD71 (BDPharmingen; M-A712; APC), anti-CD28 (BDPharmingen; L293; PE), anti-HFE (Abnova; polyclonal), anti-CD3 (Caltag; UCHT1; APC), anti-CD4 antidody (Caltag; RPA-T4; APC), anti-HLA-ABC (Dako; W6/32; PE). Post staining cells were washed twice and fixed with 2% PFA/PBS. At least 2.000 infected cells were measured with a FACS Canto II (BDBioscience). Fold modulation of cell surface receptor modulation was calculated as before [14 (link),51 (link)]. Flow cytometric measurement of FRET and the according gating strategy were performed as already described [24 (link)]. Cellular iron content was measured by the use of the fluorescent dye CA-AM which is quenched by free cellular iron [4 (link)].

Internalisation assays were performed as described in Roberts et al., 2001 (link). Briefly, cells were surface labelled at 4°C with 0.13 mg/mL NHS-SS-biotin (Pierce) in PBS for 30 min. Following surface labelling, cells were transferred to complete medium at 37°C to allow internalisation in the presence of 0.6 mM primaquine for the indicated times. Biotin was then removed from the cell surface by treatment with the cell-impermeable-reducing agent MesNa. Cells were then lysed and the quantity of biotinylated receptors determined using a capture-ELISA. The following antibodies were used for capture-ELISA: clone VC5 (BDPharmingen, Cat# 555651) for α5β1, anti-CD71 (BDPharmingen, Cat# 555534) for the TfnR, anti-HGFR (R&D Systems, Cat# AF276), and anti-EGFR1 (BDPharmingen, Cat# 555996).