Heme assay kit

The level of total heme in the blood was quantified using Heme Assay Kit (Sigma-Aldrich). Briefly, 2 μl of blood was collected using a tail snip and diluted in 100-fold ultrapure water. Heme was measured following manufacturer’s recommendations. The impact of heme metabolism on Py parasitemia was modulated using three compounds. Tin protoporphyrin (SnPP, Cayman Chemical Company) was dissolved in 25% dimethyl sulfoxide (DMSO, Thermo Fisher Scientific) and injected intra-peritoneally (IP) at 0.42 mg/mice every other day from day 0 to 13 days post Py infection. Hemopexin (Hx, Millipore Sigma) was dissolved in saline and injected IP at 10 μg/mice every other day till day from 0 to day-8 post-Py infection. Hemin (Sigam-Aldrich) was dissolved in 0.1 M NaOH and injected IP at 0.2 mg/mice every other day from 0 to 10 days post-infection.

An aqueous alkaline solution method-based Heme assay kit (Sigma-Aldrich Kft., Budapest, Hungary) was used for the heme detection. For the measurements, 3 × 10⁵ SH-SY5Y cells were treated as previously described. The method was carried out according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were collected after the treatments and lysed in 50 µL of dH₂O. The same amount of dH₂O was served as blank. The experiments were carried out on 96-well plates. For the calibration controls, 50 µL of Heme Calibrator and 50 µL of dH₂O were added into the wells, followed by the addition of 200 µL of dH₂O into the blank and the Heme Calibrator reaction wells. Next, 50 µL of each sample, then 200 µL of Heme Reagent was added into sample wells. The plate was incubated for 5 min at 25 °C. The experiments were carried out in triplicate in the four independent experiments. The diluted Heme Calibrator refers to 62.5 M heme. The converted heme form produces a colorimetric result, of which absorbance was measured by using MultiSkan GO microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 400 nm wavelength, where the OD is directly proportional to the heme concentration in the sample. The concentration of heme was expressed as µM.

The heme concentration was determined with Heme assay kit (Sigma-Aldrich Kft., Hungary), which is an aqueous alkaline solution method-based assay. The assay procedure was performed according to the manufacturers’ instructions. Briefly, after the treatments, the BV-2 cells were collected and lysed with 50 µL of water. An amount of 50 µL of water was used as a blank. An amount of 50 µL of water and 50 µL of Heme Calibrator were measured into wells of a 96-well plate, then 200 µL of water into each of the blank and Heme Calibrator reaction wells. Next, 50 µL of samples were added into wells, followed by using 200 µL of Heme Reagent added to each sample well. The plate was incubated for 5 min at room temperature. All samples and standards were run in triplicate in four independent experiments. The diluted Heme Calibrator is equivalent to 62.5 M heme. The heme is converted into a uniformly colored form, producing a colorimetric result, which can be measured at 400 nm and is directly proportional to the heme concentration in the sample. The absorbance was measured using a MultiSkan GO Microplate Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The concentration of heme was expressed as µM.

Intracellular heme was quantified in MEFs using a heme assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, 200 μL of heme reagent was added to 50 μL of sample. Subsequently, the samples were vortexed to mix. The plate containing the mixtures was incubated for 5 min at room temperature (about 22 °C), and absorbance was measured at 400 nm.

Free heme was measured in the perfusate samples before and after NMP using the Heme Assay Kit (Sigma-Aldrich, St Louis, USA) following manufacturer’s instructions.

THP-1 cells were collected after treatment by centrifugation. The heme concentration was determined using a Heme Assay Kit (Sigma-Aldrich Kft. Budapest, Hungary). The cell pellets were lysed with 100 µL of ultrapure water at room temperature for 15 min with shaking (650 rpm). Then 50 µL of each sample were mixed with 200 µL of Heme Reagent and were incubated at room temperature for 5 min. The absorbance was measured at 400 nm using a MultiSkan GO spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Heme concentration was calculated according to the instructions of the manufacturer. The heme concentration was expressed as µM. Heme concentration determinations were carried out in quadruplicate in each independent experiment.