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The temperature profile consisted of an initial step of 50��C for 2 min and 95��C for 10 min, followed by 40 cycles of 95��C for 15 sec and 60��C for 1 min. Relative quantification of the FPN1 mRNA levels was normalized to ��-actin mRNA levels and calculated with the 2?����CT method. Purified O-2A progenitor cells were cultured in confocal dishes and divided into five groups. Control group: iron efflux assay was performed on O-2A progenitor cells cultured for 3 days without any treatment. FPN1 antibody treatment group: after culturing for 2 days, cells were grown in the culture medium with FPN1 antibody (1:50) for another 24 hr. OX42 antibody treatment https://en.wikipedia.org/wiki/Pentamorphone group: after culturing for 2 days, cells were grown in the culture medium with OX42 antibody (1:50) for another 24 hr. FPN1-LV transduced group and GFP-LV transduced group as described above. For calcein loading and iron efflux assay, cultures were incubated with calcein AM (1.0 ��M final concentration) in PBS for 30 min at 37��C, followed by washing for three times with PBS to get rid of the excess extracellular calcein AM. Then cells were incubated with 100 ��M ferrous ammonium sulfate (FAS) for 20 min to increase the intracellular iron. The calcein-indicated http://www.selleckchem.com/products/LBH-589.html fluorescence was recorded at 488 nm excitation wavelengths with LSCM. After perfusing the cultures with 1 mM deferoxamine (DFO, a strong membrane-impermeant iron chelator inducing cell iron depletion), the fluorescence intensity was measured every 2 min in the next 30 min. The mean fluorescence signal of 20�C25 cells in each field was monitored. Data were the means of six independent experiments. O-2A progenitor cells were divided into five groups as iron efflux assay. Then they were incubated with 100 ��M FAS (iron loading) or DFO (iron depletion) for 24 hr. The ferritin (Ft) and transferrin receptor (TfR) protein expression in O-2A progenitor cells were http://www.selleckchem.com/products/Adriamycin.html detected by western blot as described above. The primary antibodies were mouse anti-rat Ft antibody (1:1,000) and mouse anti-rat TfR monoclonal antibody (1:1,000). Data are presented as mean �� SD. Differences were determined by Student t test or one-way ANOVA followed by a Student-Newman-Keuls test. A value of P