Bca protein analysis

Cell lysates from infected or mock infected MSCs were collected following the method of Rudolph et al [42 (link)]. Briefly, cells were washed once with cold PBS, detached by scraping, and pelleted by centrifugation before adding freeze-thaw lysis buffer containing 600 mM KCl, 20 mM Tris-Cl (pH 7.8), 20% glycerol, and 1x each of protease and phosphatase inhibitor (EMD Chemicals Inc., San Diego, CA, USA). Samples were submerged in liquid nitrogen for 5 minutes and thawed on ice for 5 minutes three times, then centrifuged at 13,200 x g for 5 minutes to pellet debris. BCA protein analysis (Pierce Biotechnology, Rockford, IL, USA) was performed on the supernatant to measure the protein concentration before use in Western blots and IDO ELISA or enzyme activity assays.

Sera samples were collected on weeks 2, 4, 6 p.i. and one week after challenge to determine induction of total IgG and neutralizing antibody response to infection. Total IgG antibody response against hMPV was determined by ELISA. Purified and filtered hMPV strain CAN97-83 was assayed for protein concentration by BCA protein analysis (Pierce Biotechnology, Inc., Rockford, IL), diluted in carbonate-bicarbonate buffer (pH 9.6) to coat 96 well plates (Maxisorb, Nunc, Denmark) with 1 µg/100 µl/well. The plates were incubated at 4°C overnight and ELISA assay was performed as described previously (37 (link)) with some modifications. After blocking the plates with 1% BSA, serum samples diluted 1:200 in PBS were added and incubated at 4°C overnight. The plates were then incubated with 1:10,000 goat anti-mouse IgG-HRP conjugate (Thermo Fisher Scientific, USA) for 1 h at 37°C. The color reaction was developed with 3,3′,5,5′-tetramethylbenzidine, TMB, (Sigma-Aldrich, USA), at OD 450 nm. Neutralizing antibodies to hMPV were measured by a plaque reduction neutralization assay, as previously described (38 (link)). The neutralizing antibody titers were defined as the reciprocal of the highest serum dilution at which ≥ 50% reduction in CPE was observed. The lowest detectable titer was 2.5 log₂. Samples with non-detectable titers were assigned a value of 2 log₂.