temperature and concentration dependent effects of mixing DFP with DFO on FO development from iron: citrate were next examined using HPLC, which allows certain identification of the FO complex when mixtures of the two chelators are employed. When DFO was incubated with iron citrate at RT for approximately 24h, FO formation was again biphasic, taking over 24h to reach completion, consistent with the spectrophotometrically established kinetics of Figure 4. The fast stage was too fast to measure by this process and had an amplitude of 3 uM FO. It could be seen that DFP improved the price of the slower second phase in a concentration Imatinib molecular weight dependent manner, together with the maximum impact at 30uM DFP. However, even low concentrations of DFP raise the price of FO creation, in keeping with the idea of DFP working as a taxi at low concentrations. As the rate of FO formation was maximally improved at 30uM DFP, a further increase in DFP concentration to 100 uM showed a tiny decrease in the rate of FO formation compared to that observed with 10 or 30 uM DFP, suggesting that DFP at higher concentrations may maintain the chelated iron and consequently slow its rate of shuttling to DFO. There is no significant difference Cellular differentiation between any of the FO concentrations measured at zero time for any mix of DFO and DFP when comparing to DFO alone. Major variations between DFO alone and DFO plus all concentrations of DFP occurred in FO formation at all future time points except wherever DFP was 3 uM. Here a significant huge difference was seen after 2 h and at all future time points. It can be seen that the rate of the next stage of FO development is temperature dependent both in the absence and presence of DFP. Ergo FO levels reach a 9. 4 uM after 8h at 37 C, while at RT this is 6. 4 uM after 8 h and only 9. 0 uM after 24h. In contrast to the gradual phase, the amplitude of FO creation in the fast phase wasn’t considerably influenced by any of the DFP levels tested. This section could not be accounted for by iron contamination in any of the reagents used, that has been established as 0. 75 uM by treatment of reaction mixtures where iron was omitted. As neither HPLC or mainstream spectrophotometry are suitable to look at the rapid phase of FO development, the rate of the faster process was examined within the first 50 seconds of reaction employing a stopped flow spectrometer. This covers the time range unavailable in the spectrophotometer and HPLC, representing the mixing and treatment time for incubations performed in these instruments. The price with this rapid phase was faster for DFP iron complex development than for DFO nevertheless the amplitude of iron chelation was similar at 50 seconds showing a similar amount of total available iron chelated by either chelator.