Victor3 multilabel plate counter spectrofluorometer

Amyloid formation was determined using the dye ThT. Thioflavin T preferentially binds to the β-sheet structures of amyloidogenic proteins/peptides. For examination of the concentration dependence of the aggregation, we incubated rTCP96 (5 μm) and LPS from E. coli (0, 10, 50, and 100 μg/ml) in buffer (10 mm Tris, pH 7.4, and 10 mm MES, pH 5.5) for 30 min at 37 °C. Moreover, rTCP96 was incubated for with 50 μg/ml of each ligand (LPS (E. coli), LPS (P. aeruginosa), lipid A (E. coli), LTA (S. aureus), and PGN (S. aureus)) for 30 min at 37 °C before measurements. Two hundred microliters of the materials were incubated with 100 μm ThT for 15 min in the dark (ThT stock was 1 mm stored in the dark at 4 °C). We measured ThT fluorescence using a VICTOR3 Multilabel Plate Counter spectrofluorometer (PerkinElmer Life Sciences) at an excitation of 450 nm, with excitation and emission slit widths of 10 nm. The baseline (10 mm Tris, pH 7.4, or 10 mm MES, pH 5.5, buffer, LPS (E. coli), LPS (P. aeruginosa), lipid A (E. coli), LTA (S. aureus), and PGN (S. aureus)) was subtracted from the signal of each sample (15 (link), 28 (link)).

The macrophage cell line RAW 264.7 (passage 4-7) in DMEM was seeded in 96-well tissue culture plates (8 × 10⁴ cells per well) overnight at 37°C in a 5% CO₂ atmosphere. AWFs were mixed with and without LPS (100 μg/ml) for 30 minutes at 37°C, which was followed by incubation with Amytracker 680 (1000× dilution from the obtained stock solution, Ebba Biotech, Lund, Sweden) for 30 minutes at the room temperature. The pre-mixed samples (20 μl) were added to the adherent RAW cells. To measure phagocytosis, the samples were incubated with the cells for 1 hour at 37°C and washed twice with DMEM media. We then measured fluorescence using a VICTOR3 Multilabel Plate Counter spectrofluorometer (PerkinElmer, USA) at excitation/emission wavelengths of 620/640 nm. The baseline uptake (of only media) was subtracted from the signal of each sample. For analysis using fluorescence microscopy, RAW 264.7 (passage 4-7) cells were seeded in 24-well tissue culture plates with inserted coverslips (2 × 10⁵ cells per well) overnight at 37°C in a 5% CO₂ atmosphere. Then cells were treated as described above. After 1 hour incubation at 37°C, cells were stained with 300 ng/ml DAPI (Sigma Aldrich, USA) for 3 min at room temperature, washed with PBS and mounted onto glass slides using a mounting media (Mountant, Permafluor, Thermo Fisher Scientific, KU) and analyzed as described above.

Amyloid formation was determined using the dye Thioflavin T (ThT). Thioflavin T preferentially binds to the β-sheet structures of amyloidogenic proteins/peptides. For examination of the concentration dependence of the aggregation, we incubated APOE (2 μM) and LPS from E. coli (100 μg/mL) in buffer (10 mM Tris, pH 7.4) for 30 min at 37 °C before measurements. Two hundred microliters of the materials were incubated with 100 μM ThT for 15 min in the dark (ThT stock was 1 mM stored in the dark at 4 °C). We measured ThT fluorescence using a VICTOR3 Multilabel Plate Counter spectrofluorometer (PerkinElmer, Boston, MA, USA) at an excitation of 450 nm, with excitation and emission slit widths of 10 nm. The baseline (10 mM Tris pH 7.4 and LPS) was subtracted from the signal of each sample [12 (link)].