Micro bca protein assay kit

6 × 10⁵ THP-1 cells were incubated with CyaA (150 ng/ml; 0.85 nM) or ET (625 ng/ml; 7.04 nM EF + 2500 ng/ml; 30.24 nM PA) for different time durations in 200 µl of DMEM with 10% FCS and no antibiotics at 37 °C. The reaction was stopped by lysing the pellet by addition of 0.2% Tween-20 in 50 mM HCl. The lysates were boiled for 10 min at 100 °C, neutralized by addition of 150 mM unbuffered imidazole, and cellular cAMP levels were measured using competitive ELISA, as described elsewhere [61 (link)]. cAMP concentrations were normalized to the total protein content, which was determined using a micro-BCA protein assay kit (Bio-Rad, Rockford, IL).

The protein entrapment efficiency in mannose-conjugated chitosan NP was estimated by an indirect method by determining difference between protein amount found in the vaccine formulation supernatant and initial amount used. The amount of protein present in the supernatant was measured using the micro-BCA protein assay kit (Biorad, USA). Entrapment efficiency (%) = [(RBD protein added − Free “unentrapped RBD protein”)/RBD protein added] * 100.