Colorimetric assay

Iron deficient chow was purchased from Harlan Laboratories (TD.99397, Indianapolis, IN) and administered to mice for eight weeks prior to transplantation. Murine serum iron measurements were made using a colorimetric assay from Thermo Scientific (Waltham, MA), which uses ferene as the complexing chromogen. Recipient B6-albino mice were sub-lethally irradiated with 5 Gy on the day of transplantation using an X-Rad 320 irradiator. They were then injected with 2×10⁵ primary CALM-AF10 leukemia cells, and followed for the onset and progression of leukemia with biweekly blood draws from the facial vein. CALM-AF10 leukemia cells co-expressing GFP were detected in the peripheral blood by flow cytometry. Mice were maintained on their pre-transplant diet, either low iron or iron replete, throughout the study. Mice in the chemotherapy experimental groups were treated with cytarabine at 33 mg/kg/day delivered intraperitoneally (IP) on days 8 – 12 after transplantation, and doxorubicin at 1 mg/kg IP on days 8-10 post-transplant. Mice in the DFX experimental groups received DFX 33 mg/kg/day on 5 of 7 days per week, beginning 2 weeks prior to transplantation and continuing until sacrificed. Mice were monitored daily for signs of distress or disease and sacrificed by CO₂ euthanasia when terminally ill. After sacrifice, spleens were removed and weighed, and the bone marrow harvested.

Cell death was estimated by lactate dehydrogenase (LDH) in media immediately after collection, using a colorimetric assay (Thermofisher, MA) and our previous procedures (Bake et al., 2016 (link)). Briefly, 50 μL of culture media was added to each well of a 96-well plate and mixed with catalyst and dye substrate mixture. After incubation for 30 min, 50 ul of stop solution was added to each well and the plate was read at 490 nm absorbance on a colorometric plate reader (Tecan, Switzerland). Calcein assay: Cell viability was determined using the Calcein-AM dye (Life Technologies, CA). After OGD, cells were incubated with calcein-AM (2.5 μm) in PBS for 20 min at 37°C and the fluorescence was measured on a plate reader (Tecan, Switzerland) with excitation/ emission set at 480 and 530 nm respectively.