96 well assay plate

Recipient cells (5 × 10⁵ cells/mL) were cultured in technical triplicates in 100 μL of fresh EV-depleted RPMI media in a 96-well assay plate (Corning Inc.; Cat#: 3610) and treated with HTLV-1 and CEM EVs as specified for each experiment. Cells were allowed to incubate for 5 days and CellTiter-Glo reagent (Promega; Cat#: G7572) was used at a 1:1 ratio to detect cell viability on a GloMax Explorer (Promega). EV-depleted RPMI media alone was cultured and used for background measurements.

Cells were cultured in technical triplicates (i.e., 1 × 10⁶ cells/mL or at 5 × 10⁵ cells/mL) in 100 μL of fresh EV-depleted RPMI media in a 96 well assay plate (Corning Inc.; Cat#: 3610) and treated with HTLV-1 EVs as specified for each experiment. Antibodies were used to treat recipient cells to test for recipient cell viability at the concentrations specified above. Cells were allowed to incubate for zero or 5 days and CellTiter-Glo reagent (Promega; Cat#: G7572) was used at a 1:1 ratio to detect cell viability on a GloMax Explorer (Promega). EV-depleted RPMI media alone was cultured and used as background.

GBM cell lines were transfected with polyQ80-luciferase (or control polyQ19-luciferase) constructs. After 24 h, cells were lysed by passive lysis buffer from the DLR Assay System and centrifuged in a refrigerated centrifuge. The cleared lysates were transfered into a fresh tube and the firefly luciferase activity of polyQ80-luciferase or polyQ19-luciferase in each group was measured. polyQ19-luciferase was used as an internal control. Here, it was designed to measure only firefly luciferase reporter activity in the treated cell lysates. Briefly, 20 μl of cell lysate prepared from polyQ80-luciferase or polyQ19-luciferase transfected cells were dispensed in triplicate into 96-well assay plate (Corning Incorporated COSTAR, NY, Washington, USA, 3925) pre-added into 100 μl of luciferase assay buffer II (containing luciferase assay substrate) from the DLR Assay System according to the manufacturer's protocol. Then the assay plate was mixed by pipetting two or three times, and stabilized luminescent signal was measured by the Veritas Microplate Luminometer (Turner Biosystems, Bio-rad, Hercules, CA, USA). Data were expressed as the ratio of polyQ80-luciferase/polyQ19-luciferase luminescence signal values in each group as described previously.^{23 (link)} All samples were assayed in triplicate, and the results were shown with three independent experiments.

Plasmids were transformed into Lemo21(DE3) cells (New England Biolabs), and grown in 96 deepwell plates overnight at 37 °C in 1 mL of LB containing 50 ug/mL of kanamycin sulfate. The next day, 100 uL of overnight cultures were used to inoculate 96 deepwell plates containing 900 uL of TBII medium (MP Biomedicals) with 50 ug/mL of kanamycin sulfate, and the cultures were grown for 2 h at 37 °C before induction with 0.1 mM IPTG. Protein expression was carried out at 37 °C for 4 h before the cells were harvested by centrifugation (4,000 x g, 5 min). Cell pellets were resuspended in 100 uL of lysis buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, 1 mg/mL lysozyme, 0.1 mg/mL DNAse I, 5 mM MgCl₂, 1 tablet/50 mL of cOmplete protease inhibitor (Roche), 0.05% v/v Tween 20), and cell were lysed by performing three freeze/thaw cycles (1 h incubations at 37 °C followed by freezing at −80 °C). The lysate was cleared by centrifugation (4,000 x g, 20 min), and the soluble fraction transferred to a 96 well assay plate (Corning, cat #3991). Concentrations of the constructs in soluble lysate were determined by sfGFP fluorescence using a calibration curve.