Bicinchoninic bca assay

Rat heart mitochondria (RHM) were isolated as described previously.²⁰ (link) Briefly, freshly excised rat hearts were homogenized in STEB buffer [250 mM sucrose, 5 mM Tris, 1 mM EGTA, 0.1% (w/v) bovine serum albumin (BSA), pH 7.4] using a Dounce homogenizer. Homogenates were centrifuged (3000 × g, 5 min, 4°C) to pellet nuclei and unbroken cells. The resulting supernatant was then centrifuged (10 000 × g, 10 min, 4°C) to pellet mitochondria. Pelleted mitochondria were resuspended in STEB buffer and recentrifuged (10 000 × g, 10 min, 4°C). The pelleted mitochondria were resuspended in STEB buffer with the BSA omitted (400 µL/heart) and the mitochondrial protein quantified by bicinchoninic (BCA) assay (Thermo Fisher Scientific).

O. dentatum larvae were isolated from infected control-fed pigs, and washed extensively in 37°C saline. The exsheathed larvae were cultured in complete media containing antibiotics and fungicide for 3 days at 37°C to obtain excretory/secretory (E/S) products. Every day the culture media was removed, stored at -80°C, and replaced with fresh media. Pooled culture media containing E/S was concentrated by centrifugation using Amicon ultra centrifugal filter units (MWCO 10 kDa, Sigma-Aldrich, Denmark), and filtered prior to testing of protein content by bicinchoninic (BCA) assay (Thermo Fisher Scientific).