Black flat bottom 96 well plate

Cattle were infected with OO L3 and venous blood samples were collected at days 0, 15 and 29 post infection. Serum was carefully harvested, without pigment contamination^{96 (link)}. Serum sample (10 µL) was added to 90 µL of PBS, followed by addition of 100 uL of Sytox Green (1:200) per well in a black 96-well flat bottom plate (Nunc) and incubated in the dark for 15 minutes at RT. Fluorescence was quantified as described in “NET Quantification” using a spectrofluorometer.

Target cells were labelled with 15 μM Calcein Green AM (Molecular Probes, Darmstadt), washed, and mixed with ex vivo expanded and stimulated PBMCs from healthy unrelated donors in RPMI 1640 GlutaMAX™ supplemented with 10 % FBS and Penicillin (100 U/mL)/Streptomycin (100 μg/mL) (Gibco, Thermo Fisher Scientific, Darmstadt) at an E : T ratio of 10 : 1. Antibody derivatives were diluted to the desired concentration with medium and added to 200 μL reaction volumes in a 96-well round bottom tissue culture plate (CellStar, Greiner bio-one). After a 3 hour incubation period at 37°C, 5 % CO₂, 100 μL supernatant was transferred to a black 96-well flat bottom plate (Nunc) and fluorescence was determined on a Berthold Mithras plate reader (Berthold Technologies, Bad Wildbad). Maximum lysis was achieved by addition of 2.5 % Triton X-100. Specific lysis was calculated as follows:
% Specific Lysis = 100 * [( RLU (sample) – RLU (background)) / (RLU (max. lysis) – RLU (background))], where RLU = relative light units and the background is the degree of lysis obtained with effector cells alone in the absence of added triplebody.

Astrocytes were plated in black 96-well flat bottom plates (Thermo Fisher Scientific Inc.). Post seeding and differentiation/adherence, cells were washed twice with PBS and preincubated with 50 µM 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Invitrogen™). Astrocytes were treated with MuVs or lmEVs (ALS: n = 3, healthy: n = 3, 85 ng of lipids). As a positive control, 100 µM H₂O₂ was added to the cells; as a negative control, 500 µM NAC (glutathione precursor and ROS scavenger) was added to the cells. Fluorescence was measured with a Biotek Multi-Mode Microplate Reader using 485 nm and 520 nm as excitation and emission filters, respectively.

GST-tagged HTT_Ex1-Q48 (10 μM) was pre-incubated in the presence or absence of DNAJB6 (0.05, 0.25, 0.5, 0.8 and 1.6 µM) or DNAJB6 LGMDD1 mutants (0.25 and 0.8 μM) for 10 min at 37 °C. All proteins in the assay were buffer exchanged into the assay buffer (50 mM HEPES pH 8.0, 200 mM KCl, and 1 mM DTT). DNAJB6 P96R mutant was unstable at pH 8.0 and the aggregation-prevention assays for this variant were performed in 50 mM HEPES pH 7.0 and 200 mM KCl buffer. Thioflavin T (ThT; Sigma) at a final concentration of 10 μM was added and the aggregation was induced by the cleavage of the GST solubility tag with 0.1 μM 3 C protease. Aggregation reactions were run for 15 h at 37 °C with continuous shaking (500 rpm) and monitored by ThT fluorescence (excitation = 440 nm, emission = 485 nm), using an area scan mode with a 3 × 3 matrix for each well. Black, flat-bottom, 96-well plates (Nunc) sealed with optical adhesive film (Applied Biosystems) were used. The experiments were conducted in triplicate and the mean ± standard deviation is reported.

Chloromethyl‐2′7′‐dichlorodihydrofluorescein diacetate (CM‐H₂CFDA) (Molecular probes, Life Technologies, Carlsbad, CA, USA) was used to detect the production of cytosolic reactive oxygen species (cROS). A total of 1 × 10⁶ neutrophils in 1 mL were incubated in 24‐well plates for 30 minutes with CM‐H2CFDA in RPMI plus 2% autologous plasma with 25 ng/mL GM‐CSF at 37°C and 5% CO_2. Cells were harvested, washed with PBS, and stimulated for 30 minutes with the indicated stimuli in HBSS at 37°C and 5% CO₂. Immediately thereafter, fluorescence was assessed by flow cytometry on a BD FACS Canto II and analyzed with FlowJo software.
MitoSOX Red (Life Technologies) was used to detect the mitochondrial superoxide production in a plate reader assay. A total of 2 × 10⁵ neutrophils in 200 µL were seeded into black flat bottom 96‐well plates (Thermo Fisher) in HBSS medium containing 25 ng/mL GM‐CSF and preincubated for 1 hour with 5 µM MitoSOX Red at 37°C and 5% CO₂ and then, different stimuli added. Induction of fluorescence by mROS was detected at 2 minutes intervals with a TECAN Infinite M1000 plate reader. The area under the curve was calculated and compared to the medium control.