Multi element calibration standard 2a

Prior to metal and heme analyses, frozen tissues were added to three volumes of pure water and homogenized in ceramic bead tubes (Qiagen) using an Omni Bead Ruptor 24. For metal analysis, homogenate aliquots were digested overnight in 5:1 HNO₃:H₂O₂, dried, and resuspended in 2% HNO₃ for analysis using an Agilent 7900 ICP-MS. Calibration standard solutions for determination of Fe, Zn, Cu and Mn were prepared from Agilent multi-element calibration standard-2A. Protein concentrations of homogenates were determined by BCA Protein Assay (Thermo Fisher Scientific) for normalization.

Synchronized L1 stage worms were grown on NGM plates seeded with OP50 until reaching the L4 stage. Worms were washed extensively with M9 buffer and incubated in M9 buffer at room temperature for 2 hr to allow for purging of the gut followed by three rinses with ddH₂O. Worms were pelleted and dried, and metal analysis determined by inductively-coupled plasma-optical emission spectroscopy (ICP-OES) (Children’s Hospital Oakland Research Institute (CHORI) Elemental Analysis Facility). Empty tubes were run in parallel to serve as controls. Iron content was normalized to sulfur content. For pathogen experiments, worms were harvested in water, washed and homogenized for metal analysis. Protein concentration in lysates was measured using the BCA Protein Assay (Thermo Fisher Scientific) and 100 µg of lysate was digested overnight in 5:1 HNO₃:H₂O₂, dried, and the pellet resuspended in 2% HNO₃ for metal analysis by inductively-coupled plasma mass spectrometry (ICP-MS) (University of Utah, Center for Iron and Hematology Disorders, Iron and Heme Core). At least 3–5 biological experiments were performed for each strain with triplicate technical replicates. Calibration standard solutions for determination of Fe, Zn, Cu and Mn were prepared from Agilent multi-element calibration standard-2A. Iron content was normalized to protein.

Nitric acid (69%, part# 100441) was purchased from Merck Millipore (Darmstadt, Ger-many). Deionized water (DIW, 18.2MΩ·cm) was obtained from a Milli-Q system (Millipore, MA, USA). Multi-element calibration standard 2A (part# 8500-6940) and 4 (part# 8500-6942), environmental calibration standard (part# 5183-4688), and standard solutions of scandium (Sc, part# 5190-8578) and rhodium (Rh, part# 8500-6945) were purchased from Agilent Technologies (Santa Clara, CA, USA). The Standard Reference Material (SRM) of rice flour (1568b) was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA).