Pierce protein bca protein assay

Following 24 h incubation with the bread digestions, the cellular medium was aspirated, the cells were rinsed with 18 Ω MilliQ H₂O and subsequently lysed by scraping in 200 μl of Cellytic M (Sigma-Aldrich, UK). Cell pellets were kept on ice for 15 min and stored at −80 °C. For analysis, samples were thawed and centrifuged at 14,000g for 15 min. Cellular debris was discarded and the supernatant containing the proteins was used for ferritin determination using the Spectro Ferritin ELISA assay (RAMCO, USA). The ferritin concentration in the samples was determined using a microplate reader at an excitation wavelength of 500 nm according to the manufacturer’s protocol. Ferritin concentrations were normalised to total cell protein using the Pierce Protein BCA protein assay (ThermoFisher Scientific, UK).

RASMC were seeded at 300,000 cells/well in fibronectin-coated 6-well plates. Supplemented media was replaced by serum-free media 24 h prior to experiment commencement. Cells were treated with media only (untreated control), 10 µM treatment, 0.02% DMSO (vehicle control), and incubated for 24 h at 37 °C, 5% CO₂, in a humidified atmosphere. Cells were washed 3x with PBS and cells lysed with Extraction Reagent Buffer (Enzo Lifesciences, City, UK) and stored at −80 °C until required, undergoing one freeze-thaw cycle. HO-1 protein in cell lysates was determined by use of Rat Hmox-1 ELISA Kits (Enzo Lifesciences, Exeter, UK) according to the manufacturer’s instructions. HO-1 protein concentrations were normalised to the total cell protein content using the Pierce Protein BCA protein assay (Thermo Fisher Scientific, Loughborough, UK).

Ferritin formation was measured 24 h after treatment. Cells were rinsed with Milli-Q (18.2 MΩ) H₂O and subsequently lysed by scraping in 100 µL (12-well plates) or 200 µL (6-well plates) of CelLytic M (Sigma-Aldrich, Gillingham, UK). Cell lysates were kept on ice for 15 min and stored at −80 °C. For analysis, samples were thawed and centrifuged at 14,000× g for 15 min. Cellular debris was discarded and the supernatant containing the proteins was analysed for ferritin using the Spectro Ferritin ELISA assay (Ramco Laboratories Inc., Stafford, TX, USA). The ferritin concentration in the samples was determined using a microplate reader at an excitation wavelength of 500 nm according to the manufacturer’s protocol. Ferritin concentrations were normalized to total cell protein using the Pierce Protein BCA protein assay (ThermoFisher Scientific, Loughborough, UK).