Polarstar omega plater reader

Synthetic PSMα3 peptide (MEFVAKLFKFFKDLLGKFLGNN) was purchased from Biosynthesis. PSMα3 (2.5 µg/ml) was fibrillized in the presence or absence of CpG DNA (In vivogen, ODN 1826) at a charge ratio of 20. Fibrillization was monitored in the presence of 10 µM Thioflavin T (Sigma, T3516) for 10 minutes protected from light. The relative fluorescent units (RFU) were detected (excitation 440 nm/emission 490 nm) using a BMG Labtech POLARstar Omega plater reader.

Synthetic peptides that correspond to the fourth and fifth repeats of CsgA, CsgA_R4-5, and CsgA_R5-4N122A described previously^{42 (link)} were synthesized by Biosynthesis Inc. Polymerization of the synthetic peptides was previously described^{43 (link)}. To monitor the fibrillization of the synthetic CsgA peptide, 100 μM CsgA_R4-5, or 100 μM CsgA_R5-4N122A was mixed with an equal volume 10 μM ThT in the presence or absence of 0.5 mg/ml mAb 3H3 or control antibody 6A in a black Nunc 96-well plate (Fisher). The plate was sealed with an optical plate cover (Eppendorf). Fluorescence of ThT (excitation 440 nm/emission 490 nm) was monitored at 37 °C using a BMG Labtech POLARstar Omega plater reader. Readings were taken at 8-min intervals for 36 h. Lag time (t₀) was calculated using the following formula: t₀ = t_1/2 − 2t_e, where t_1/2 is the time required to reach half the maximum fluorescence intensity and t_e is the elongation time^{43 (link)}.