Hemin chloride

Hemin chloride (product number 51280), protoporphyrin IX disodium salt (product number 258385), and sodium dithionite (product number 71699) were purchased from Sigma Aldrich (Darmstadt, Germany). Ferric mesoporphyrin IX chloride (mesoHemin chloride, product number sc-396889) was purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Millipore water was used for LC-MS analysis (from an in-house Millipore water purification system, Darmstadt, Germany). LC-MS-grade methanol (product number 34966) was purchased from Honeywell (Offenbach/Main, Germany). Formic acid (LC-MS grade, product number 84865.180) and hydrochloric acid (37%, product number 20252.290) were purchased from VWR Chemicals (Darmstadt, Germany). Analytical-grade sodium hydroxide (product number 1375.1000) and ammonia solution (25% in water, product number 2672.1011) were purchased from Chemsolute (Renningen, Germany). Hydrogen peroxide (30% in water, product number AB129030) was purchased from abcr (Karlsruhe, Germany). Ammonium acetate (LC-MS grade, product number 73594) was purchased from Merck (Darmstadt, Germany).

This bioassay was performed to evaluate the inhibition of FP biocrystallization in presence of the essential oils and the pure compounds [22 (link)]. The bioassay was carried out in a non-sterile 96-well plate flat-bottom at 37 °C for 18–24 h. The EOs and pure compounds were tested at several concentrations (EOs at 10, 5, and 2.5 mg/mL; and compounds at 100, 50, and 25 µg/mL). Chloroquine biphosphate (Sigma-Aldrich) was used as the reference drug. A series of solutions were added to the plate, namely: 0.5 mg/mL of hemin chloride (Sigma-Aldrich) freshly dissolved in dimethylsulphoxide (DMSO) (50 µL), 100 µL of 0.5 M sodium acetate buffer (pH 4.4), and 50 µL of EOs or compounds dissolved in DMSO. After 18–24 h, the plates were centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. The remaining pellet was resuspended in 200 µL of DMSO so as to remove the unreacted FP. The plate was centrifuged again and the supernatant was discarded. The pellet was dissolved in 150 µL of 0.1 M NaOH, and the absorbance measured at 405 nm. The percentage of inhibition of the FP biocrystallization was calculated as follows:

Proteins were desalted with a C4 ZipTip (MilliporeSigma) and so prepared to achieve concentrations that ranged from 2 to 10 mg/ml. For matrix-assisted laser desorption ionization time of flight (MALDI TOF) mass spectrometry, a matrix solution composed of 10 mg/ml α-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.1% trifluoroacetic acid (v/v) was made. One µL of the CHCA matrix was mixed with 1 µL of protein and allowed to dry at 30°C for 15 min. Approximately 1000 laser shots were collected for each sample on a Bruker Ultraflextreme MALDI TOF/TOF mass spectrometer with positive ion setting in the reflector mode. Hemin chloride (Sigma) was used as standard.

Staphylococcus aureus Oxford, Escherichia coli DH5α, Streptococcus mutans UA159, Streptococcus mutans ATCC 10449, Streptococcus sobrinus OMZ 176, Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, Fusobacterium nucleatum ATCC: 10953, 25586, 33568, 49256; Porphyromonas gingivalis ATCC 33277, and Prevotella intermedia ATCC 25611 were from the University of Otago culture collection. Staph. aureus and E. coli were included as reference gram-positive and gram-negative bacteria, respectively [27 (link), 28 (link)].
Staph. aureus and E. coli were cultured in tryptic soy broth (TSB) (BactoTM, Becton Dickinson Co., Sparks, MD, USA) and on TSB agar, under aerobic conditions. Streptococci were cultured on Columbia sheep-blood agar (CBA) (Fort Richard, Mt. Wellington, New Zealand) under anaerobic conditions (10% H₂, 5% CO₂, and 85% N₂) in a MACS work station (MG500, Don Whitley Scientific Ltd., Shipley, United Kingdom) and were grown in brain heart infusion (BHI) (Difco Laboratories, Detroit, MI, USA). F. nucleatum, P. gingivalis, and P. intermedia were cultured and maintained under anaerobic conditions on CBA and in prereduced BHI supplemented with hemin chloride (5 μg/ml; Sigma Chemical Co., St. Louis, MO, USA) and menadione (1 μg/ml; Sigma). All incubations were at 37°C.

To study proliferation, B cells were labeled with 1 μM DDAO (Thermo) and cultured in full RPMI supplemented with 4 μg/ml CD40L (R&D systems), 100 ng/ml BAFF (PeproTech), 1 μg/ml anti‐IgM (Jackson Immunoresearch), or 2.5 μg/ml CpG (Invitrogen). DDAO dilution (and mCherry expression in CRISPR‐targeted B cells) was detected by flow cytometry 3 days post‐culture.
For cell expansion studies, full RPMI was supplemented with one of the following: 5 μM hemin chloride (Sigma), 50 μg/ml ferrous sulfate (Sigma), 10 g/l d‐Glucose (Gibco), 10 mM l‐Glutamine (Gibco), or 5× non‐essential amino acids (NEAA, Gibco).

The ability of the 5-phenoxy primaquine analogs to inhibit hematin polymerization was investigated using a protocol modified from the one described previously [32 (link)]. Briefly, hemin chloride (30 μM; Sigma) was dispensed in a 96-well plate, followed by the addition of the compounds (1-400 μM solution in water), and the volume was adjusted to 200 μL with phosphate buffer pH 5. After it was left standing for 15 min, Tween 20 (0.5 μM; Sigma) was added. After incubation at 37 ^oC for 1 h, the absorbance was measured at 405 nm. The assay was performed in triplicate, and results were expressed as the percentage of inhibition relative to hemozoin formation in a negative control. The IC₅₀ values were obtained from the sigmoidal dose–response curves using nonlinear regression curve fitting analyses with GraphPad Prism version 3.00 software. Each IC₅₀ value is the result of at least three separate experiments.