Black walled 96 well microtiter plates

The switch-off Pseudomonas putida BS566::luxCDABE bioreporter in 96-well plate assay format was used as previously reported, with minor modifications [18 (link)]. Bacteria were pre-cultured overnight at 28 ± 2 °C under shaking conditions (140 rpm) in artificial wastewater (AW) then freshly diluted in order to reach a final concentration per well of 10⁸ CFU·mL⁻¹. Stock suspensions of Ag NPs were freshly prepared at 222 mg·L⁻¹ (i.e., corresponding to 200 mg·L⁻¹ final when used at 90% (v/v)) in collected wastewater samples prior to each experiment (i.e., via weighting of NPs) then further serial diluted to give final tested concentrations of 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 mg·L⁻¹. All wastewater samples were supplemented with D-glucose (0.5%, w/v) prior to use in order to ensure a consistent minimal amount of carbon source. Assays were conducted with 90 µL CW or FW (spiked with Ag NPs) mixed with 10 µL AW (containing the P. putida bioreporter) in black walled 96-well microtiter plates (Greiner bio-one, Germany). Monitoring of the emitted luminescence evolution was performed using a SpectraMax M5 reader (Molecular Devices, Sunnyvale, CA, USA) in a kinetic mode for 2 h at 28 ± 2 °C. Results were expressed in Relative Luminescence (% RLU) and plotted against time (min) for selected conditions. Ag NP toxicity was expressed as IC₅₀ (mg·L⁻¹) as derived at 1 h.

Monomeric human α-Syn peptide at 100 µM was incubated in PBS (pH 7.4) with 25 µM thioflavin-T (ThT) in the presence or absence of various seeding agents including BSA-conjugated cyclized and linear peptide epitopes (100 nM), sonicated PFFs (10 nM), oligomers (100 nM) and physiologic tetramers (100 nM) in a 120 µL reaction volume per well of black-walled 96-well microtiter plates (Greiner Bio-One, Monroe, NC, USA). In studies of neutralizing activity, mAb was added at 0.1 nM. Plates were incubated at 37 °C with shaking for 30 sec prior to each hourly reading of ThT fluorescence (excitation at 440 nm, emission at 486 nm) using a Wallac Victor3v 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA). To be noted, in some studies, continuous shaking was used to promote aggregation, giving rise to higher levels of aggregation with monomers alone.

Target cells were stained with 10 μM calcein‐AM (Thermo Scientific, Germany) for 30 minutes. Then, 1 × 10⁴ cells per well were placed in a 96‐well flat‐bottom microtiter plate with CAR T cells at effector‐to‐target cell ratios of 40:1 to 10:1 or alone (spontaneous release). After 4 hours, the supernatant was transferred into black‐walled 96‐well microtiter plates (Greiner Bio‐One, Germany) and fluorescence was quantified with a microplate reader GloMax Discover (Promega, Germany). To determine maximum release, cells were lysed with 9% Triton X‐100. Data were expressed as Arbitrary Fluorescence Units (AFU) and specific lysis was calculated by [(test release − spontaneous release)/(maximum release − spontaneous release)] × 100.