, 1995). The cell monolayer was detached using a plastic spatula, followed by centrifugation and collection of the supernatant (representing cell surface-bound 59Fe-Tf) (Richardson et al., 1995). The cell pellet was re-suspended in PBS (1 mL), and 59Fe levels were quantified in the pellet and supernatant using a gamma counter (2480 selleckchem Palbociclib Wizard2, Perkin Elmer, Turku, Finland). Data are presented as a percentage in comparison with control cells. Cellular 59Fe efflux from cells by iron chelators after pre-labelling with 59Fe-Tf Cells were plated as in uptake experiments and pre-labelled for 3 h/37��C with 59Fe using 1 mL of media per plate containing 59Fe-Tf (60 ��g?mL?1), implementing standard methods (Richardson et al., 1995). The cell monolayer was then washed four times on ice with ice-cold PBS (Richardson et al.
, 1995). Cells were then re-incubated for 3 h/37��C with medium alone or medium containing the chelators (1�C20 ��M). The media was removed, and the amount of 59Fe in the media and monolayer was assessed as above. Results were expressed as the percentage of 59Fe mobilized from cells incubated with control medium alone. qRT-PCR qRT-PCR was performed as described previously (Le and Richardson, 2002; Boult et al., 2008). All reactions were performed using human 18S ribosomal RNA as an internal standard (Life Technologies Ltd, Paisley, Renfrewshire, UK) and contained both human probe and primers to TfR1, ferritin-H and/or ferroportin (FPN). Western blotting Western blotting was performed by standard methods[30] using antibodies to TfR1 (Cat. #: ab1086 Abcam, UK; 1:500), FPN (Cat.
#: ab85370 Abcam, UK; 1:300) or ferritin heavy chain (ferritin-H; Cat. #: ab65080 Abcam, UK; 1:500). To ensure normalization of protein loading, ��-actin (Cat. #: ab8226 Abcam, UK; 1:2000) monoclonal antibody was employed (Yuan et al., 2004). Immunoreactive bands were subjected to densitometry using NIH Image 1.62 software (National Institutes of Health, Bethesda, MD). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cellular proliferation assay Cells (1 �� 106?mL?1) were plated into 96-well plates and upon attaining 70% confluence were incubated with media (with or without chelator) for 24�C48 h. At the end of the culture period, 10 ��L of MTT solution (5 mg?mL?1 in PBS) was added to each 100 ��L of culture media and incubated for 3 h/37��C.
The medium was then aspirated, and the cells were solubilized using DMSO (100 ��L). The absorbance at 490 nm was read using a Bio-Tek ELx800 absorbance microplate reader (Potton, Bedfordshire, UK), and the results were expressed as percentage viability with respect to the untreated control. BrdU proliferation assay The BrdU assay was performed according to manufacturer’s Brefeldin_A instructions (Roche Applied Science, Indianapolis, IN).