M3 basea

ROS was detected using a 2′-7′-dichlorodihydrofluorescein diacetate (DCFDA) - cellular ROS detection assay kit from Abcam (Cambridge, MA). Briefly, NCM460 and SW620 cells (2.5 × 10⁴/100 μl) were plated in Costar 96-well black solid-bottom culture plates (Corning, Corning, NY) and incubated overnight at 37°C. Complete media was replaced with starvation media (M3:BaseA (InCell Corp.) +FBS (0.5%), +PenSptrep (0.5%)) then the cells were incubated overnight at 37°C. Cell were washed with 1 x PBS and incubated with 25 μM DCFDA for 45 m at 37°C. After incubation, 100 μl/well of the various test compounds (Hb(n) or Hb) at various concentrations were added to the wells. Appropriate blanks (with, without DCFDA) were used to correct for background autofluorescence of controls, and cells treated with 50 μM tert-butyl hydroperoxide and DCFDA were used as positive controls. Fluorescence was measured at 1 h intervals for up to 24 h (data shown represents 0 h, 2 h, 6 h, 12 h, and 24 h only) in a BioTek Synergy 2 plate reader (BioTek Instruments) using excitation and emission wavelengths of 485 nm and 535 nm. Fluorescence is presented as a percent of control values by the formula [Ftexp/Ftcontrol], where Ftexp = fluorescence at any time after treatment in a given well and Ftcontrol = mean fluorescence of the respective untreated control replicates.

Human Hb and Hb alpha and beta chains and hemin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The human NCM460 cell-line (normal colon epithleial cells) was obtained from INCELL Corporation Inc. (San Antonio, TX, USA). Some studies also used a human colon adenocarcinoma cell line (SW620) obtained from primary and secondary tumors from a 51 years old Caucasian patient.^{30 (link)} Cells were grown in proprietary M3^TH Base complete medium supplemented with Fetal Bovine Serum (FBS) (Invitrogen, Grand Island, NY). They were plated at a density of 1 × 10⁵ cells/ml in 6-well dishes and allowed to grow overnight. The media was then replaced with normal media (M3:BaseA (InCell Corp.) +10% FBS (Atlanta Biologocals) +1% PenStrep (Life Technologies)) or normal media containing different concentrations of test compounds for different period of times.