α-Syn fibril formation was monitored by Th-S binding. Th-S is a fluorescent dye that interacts with fibrils in a β-sheet structure. Each sample (10 μl) was mixed with 40 μl of Th-S (25μM) in PBS. Fluorescence was measured in a 384-well, non-treated, black micro-well plate (Nunc, Denmark) using a Victor X3 2030 (Perkin Elmer) microplate reader with excitation and emission wavelengths of 450 and 486 nm, respectively. To allow for background fluorescence, the fluorescence intensity of a blank well containing only PBS solution was subtracted from all readings.
Black microwell plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
The Black Microwell Plate is a laboratory equipment item used for various applications in scientific research and analysis. The plate features a dark-colored surface and individual wells designed to hold small volumes of samples or reagents. This type of plate is commonly used in various assays, optical measurements, and high-throughput screening procedures.
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6 protocols using black microwell plate
1
Monitoring α-Synuclein Fibril Formation
2
Cell Fluorescence Quantification in Microwell
3
Modulation of α-Synuclein Aggregation
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α‐Syn seeds (1 µm ) in PBS pH 7.4 were incubated alone or in combination with Nbα‐syn01 (8 and 10 µm ) or BivNbα‐syn01 (2 and 4 µm ), in sealed 1.5 mL sterile polypropylene tubes for 1 h at 37 °C with continuous shaking at 300 r.p.m. (total volume = 100 µL). α‐Syn monomers (25 µm ) were then added in PBS to a total volume of 500 µL, and the reaction mixture was further incubated at 37 °C with continuous shaking at 800 r.p.m. Protein sample aliquots were taken out at 0, 2, 4 and 6 h intervals and immediately flash‐frozen in liquid nitrogen. α‐Syn aggregation was determined using Th‐S fluorescence assay. Each sample (10 µL) was mixed with Th‐S reagent (40 µL–25 µm ) in PBS. Resultant fluorescence was detected in a 384‐well, non‐treated, black microwell plate (Nunc) with a Perkin Elmer EnVision Multimode Plate Reader using 450 and 486 nm excitation and emission wavelengths respectively.
4
Cell Fluorescence Measurement Protocol
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An aliquot of 5 × 106 cells in a volume of 200 μL PBS was pipetted in a black microwell plate (Nunc). The fluorescence was then measured in an Anthos Zenyth 3100 (Anthos Labtec Instruments) plate reader with an excitation wavelength of 485 nm and an emission wavelength of 595 nm.
5
Amyloid Aggregation Kinetics Modulation
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α-syn seeds were incubated in PBS either alone (1 µM), or in combination with scFv-pF (40 µM), scFv-pC (8 µM) or Syn-F2 (1 µM), in sealed 1.5 mL sterile polypropylene tubes for 1 hour at 37 °C with continuous shaking at 300 rpm in a total volume of 100 µL. 25 µM α-syn monomers were added in PBS at a final volume of 500 µL, and the reaction mixture was incubated at 37 °C with continuous shaking at 300 rpm. Aliquots were collected at 1 hour intervals between 0–6 hours and were flash frozen using liquid nitrogen. The aggregation of α-syn was assessed using Th-S fluorescence assay. 10 µL of each sample was mixed with 40 µL of Th-S reagent (25 µM) in PBS. Fluorescence was measured in a 384-well, non-treated, black micro-well plate (Nunc) using a Perkin Elmer EnVision Multimode Plate Reader with excitation and emission wavelengths of 450 nm and 486 nm, respectively.
6
In-vitro α-Synuclein Seeded Aggregation Assay
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In‐vitro a‐syn seeded aggregation assay was carried out as previously described (Hmila et al., 2022 (link)). Briefly, α‐syn pure seeds (2 μm) in PBS pH 7.4 were incubated alone or in combination with 10 μm of either Nbα‐syn01, ∆NterNbα‐syn01 or NbS21A in sealed 1.5 mL sterile polypropylene tubes for 1 h at 37 °C with continuous shaking at 300 RPM. (total volume = 100 μL). The incubated samples were then added to tubes containing α‐syn monomers in PBS to a get a final concentration of 25 μM in a total volume of 500 μL, and the reaction mixture was further incubated at 37°C with continuous shaking at 800 RPM. Aliquots were taken out at 0, 2, 4, 6, and 24 h intervals and immediately flash‐frozen in liquid nitrogen. α‐Syn aggregation was determined using Th‐S fluorescence assay. Each sample (10 μL) was mixed with Th‐S reagent (40 μL–25 μm) in PBS. Resultant fluorescence was detected in a 384‐well, non‐treated, black microwell plate (Nunc) with a Perkin Elmer EnVision Multimode Plate Reader using 450 and 486 nm excitation and emission wavelengths, respectively.
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