Biomek i5

Lipidomic analysis was completed using an established method previously described [49 (link)], resulting in the quantification of 856 lipid species from 10 μL of rat plasma. Sample extraction was completed using a Biomek i5 sample automation system (Beckman Coulter, Mount Waverley, VIC Australia). To each 10 μL plasma sample, 90 μL of isopropanol (Fisher Scientific, Malaga, Western Australia) containing stable isotope-labelled internal standards (LipidyzerTM Internal Standards Kit from Sciex, Framingham, MA, USA, and SPLASH LipidoMIXTM, Lyso PI 17:1, Lyso PG 17:1, and Lyso PS 17:1, Avanti Polar Lipids, Alabaster, AL, USA) was added, and then mixed for 20 min. Samples were then centrifuged (3500× g for 10 min at 4 °C). Supernatant aliquots of 70 μL were then transferred into an Eppendorf 350 μL 96-well plate for LC-MS analysis. Samples were analysed within 24 h of preparation. QC samples were prepared using a commercial pooled human plasma sample (BioIVT, New York, NY, USA) and underwent replicate extraction (n = 5) using the method described. These replicate samples were periodically analysed throughout the analytical sequence.

Neutralization assays were carried out essentially as described previously (18 (link)). Serum antibodies were diluted 4-fold in negative serum and 10-point 3-fold titrations in 25% negative serum were performed in 384 well polystyrene plates in duplicate using a Beckman (Biomek i5) liquid handler. Positive and negative control antibodies and an unrelated control (hIgG1 isotype) were tested in a 10-point, 3-fold serial dilution starting at 8 µg/mL, 2 µg/mL and 8 µg/mL, respectively, in 25% negative serum. An empirically pre-determined fixed amount of pseudovirus (Wuhan, E484Q, E484K, or the B.1.351 spike) was dispensed by WDII liquid dispenser on titrated serum antibodies and controls and pre-incubated for 20 minutes at 37°C. Following pre-incubation, the virus-antibody complexes were transferred by Biomek i5 to 8,000/well VeroE6 cells in white, opaque, tissue culture treated 384W plates, and incubated for 16-20 hours at 37°C. Control wells included virus only (no antibody; 14 replicates) and cells only (14 replicates). Following infection, cells were lysed with Promega BrightGlo and luciferase activity was measured on the Biotek Synergy Neo2 Multimode Reader.

Samples were kept frozen during the entire procedure preceding RNA extraction using dry ice and liquid nitrogen to flash freeze. Samples were pulverized using Cp02 cryoPREP Automated Dry Pulverizer (Covaris, 500001, Covaris, Woburn, MA) for SVV- and CVA21-treated tumor samples. For SVV samples, 10 mg of pulverized sample was weighed and transferred to a 2 mL microcentrifuge tube (Sample Tube RB, QIAGEN 990381, Hilden, Germany). Buffer RLT Plus with B-mercaptoethanol (600 µL) was added to each sample and lysed. The remaining steps were performed using the QIAcube (QIAGEN, Hilden, Germany), following the manufacturer’s protocol for QIAGEN RNeasy Plus Mini Kit (QIAGEN 74134) under the section for Purification of Total RNA from Animal Tissues. RNA samples were treated with DNAseI (RNAse-free) (New England Biolabs, #M0303S, Ipswich, MA) after extraction.
For CVA21 samples, 10 mg of pulverized sample was weighed and transferred to a 1.5 mL microcentrifuge tube. Lysis Buffer/Proteinase K mixture (400 µL) (RNAdvance Tissue Kit, Beckman Coulter, A32649, Pasadena, CA) was added to each sample. Samples were incubated at 37 °C for at least 30 min to lyse samples completely. The remaining steps were performed using the Biomek i5 (Beckman Coulter, B87583, Pasadena, CA) following manufacturer’s protocol (RNAdvance Tissue Kit). The RNAdvance Tissue Kit includes a DNase I treatment step.