Echo 525

Clinical material (viral transport medium from throat/nose swabs), provided for validation by NWLP, included samples left over after clinical diagnosis as per standard practice for the validation of new assays and platforms. Patient samples were stored at room temperature for no more than 48 h after the initial analysis by NWLP before they were purified and analysed on our platform. Results (Ct values) were compared directly with those obtained by NWLP. As NWLP uses a nested PCR method, Ct values were reported as being the summation of the first and second PCR steps.
qPCR reactions were set up using the TaqPath 1-Step RT-qPCR Master Mix, CG kit, and the CDC N1 Primers according to the manufacturer’s instructions and thermocycling settings (according to the CDC protocol). Final reaction volumes were 10 µL with 5 µL of extracted RNA template. Liquid transfer of the qPCR master mix was performed using an Echo 525 (Labcyte) from a 6-well reservoir (Labcyte). Extracted RNA templates were transferred using a multichannel pipette. Plates were sealed with MicroAmp Optical Adhesive Films (Thermo Fisher Scientific) and spun at 500 × g in a centrifuge. An Analytik Jena qTower³ auto was used for thermocyling and measurements were taken in the FAM channel.

Reactions were studied in 384 or 1536 well microtiter plates (Greiner), at a volume of 10 μL or 2 μL, respectively, and prepared using an Echo® 525 acoustic liquid handling robot (LabCyte). A general Python script for robot transfer instructions is available at https://github.com/jmacdona/ODE_MCMC_tools. Liquid droplets were transferred as multiples of 25 nL to a final volume of 2–10 μL as technical triplicate replicates. Plates were sealed with Breathe‐Easy^® sealing membrane (Sigma) and briefly centrifuged at 1000 × g for 10 s. A CLARIOstar plate reader (BMG Labtech, Germany) was used for enzyme incubations and kinetic fluorescence measurements using monochromator system optics. Standard measurement settings were 30°C incubation temperature, 40 flashes per well, 10 s of 300 rpm orbital shaking prior to measurement, 0.1 s settling time, 1 min recordings for 180 cycles, 425‐10 nm excitation, 520‐20 nm emission and 1600 gain.

The chemical screening was performed essentially as described previously (Du et al. 2022 (link)). Briefly, P3 NRVMs were isolated and cultured on 96-well plates at 15,000 cells per well. After 48 h, the culture medium was aspirated, and fresh culture medium (high-glucose DMEM containing 200 μΜ H₂O₂, 0.5% FBS, and 1% penicillin–streptomycin) with 2 μM small molecules was immediately applied. With regard to the preparation of 2 μM small-molecule plates, the chemical library arrayed in 384-well microplates consisted of 3,142 FDA-approved compounds (HY-L035, MedChemExpress, China); the compounds were transferred in a volume of 20 nL (10 mM) to 96-well plates using Echo 525 (Labcyte); and culture medium (100 μl DMEM containing 200 μM H₂O₂, 0.5% FBS, and 1% penicillin–streptomycin) was then distributed to each well using Multidrop Combi (Thermo Scientific), resulting in a final working concentration of 2 μM compounds. Both negative control (0.5% FBS) and positive control (10% FBS) were treated with 2 μM DMSO instead of compounds, while only the negative control was treated with H₂O₂. All of the NRVMs were then cultured for 24 h before LDH detection.

qPCR analysis of both
loxTags and PCRTags was done by adapting an earlier established protocol.^{15 (link)} The methodology described here represents the
optimized procedure after the initial loxTag screen. LoxTag and PCRTag
primer pairs were dispensed using an Echo525 instrument (Labcyte).
25 nL of 50 μM premixed forward and reverse primers was dispensed
in 384 PCR plates (Sarstedt, 72.1984.202); 25 nL was not included
in the total volume calculation. Primer plates were prepared in bulk
and stored for up to 4 weeks. To perform the 1 μL qPCR assay,
1× Luna Universal qPCR Master Mix (M3003, NEB) containing 2 ng/μL
template DNA was dispensed using a nanoliter dispenser (Cobra 4-channel
dispenser, ARI). Plates were spun down at 500 × g for 5 min followed by sealing at 180 °C for 2 s with optical
clear permanent seal (Agilent, 24212-001) using the Plateloc Thermal
Microplate Sealer (Agilent). qPCRs were run using Applied Biosystems
QuantStudio 5. Samples were preincubated at 50 °C (1.6 °C/s)
for 2 min followed by 1 min 95 °C (2.57 °C/s). A two-step
PCR reaction was performed with 30 cycles each 95 °C (2.57 °C/s)
1 s and 67 °C (2 °C/s) 1 min, with single acquisition. Melting
curve was acquired from 97 °C (0.1 °C/s) with continuous
acquisition. Raw data were exported and analyzed by using a custom
R script applying ggPlot2 for heatmap generation.

Experiments were designed and randomized using SAS JMP and Riffyn. Colorimetric LAMP reactions (NEB WarmStart® Colorimetric LAMP 2× Master Mix) were performed with a lower final reaction volume of 5 µL. Master Mix, primers, and template were transferred to a 384-well small volume LoBase plate (Greiner Bio-One) using an Echo 525 (Labcyte). The plate was then sealed with a MicroAmp Optical Adhesive Film (Thermo Fisher Scientific) and centrifuged for 1 min at 500 g. The plate was incubated at 65 °C in a CLARIOstar Plus (BMG Labtech) plate reader and absorbance measurements were taken at 415 nm every minute for 60 min. Double orbital shaking of 600 r.p.m. for 30 s was performed before the first, sixth, and eleventh cycles.