Spectramax id3 microplate reader

Acceptor cells were seeded 24 h before the uptake experiment, at 20,000 cells per well in a 96-well plate. NLuc-Hsp70 EV input was added in serum-free DMEM for a final concentration of 1–10 μg protein/mL, or as specifically indicated. Cells were incubated with EVs at 4 or 37 °C for 0–24 h, or the specifically indicated time. After incubation, cells were washed with PBS and then lysed in the aforementioned lysis buffer, prior to transferring in white 96-well plates. Finally, 50 μL of Nano-Glo^TM reagent (Promega, Wisconsin, USA) was added on each well and luminescence activity was read using iD3 SpectraMax microplate reader (Molecular Devices, California, USA) or Centro LB 960 microplate luminometer (Berthold, Germany) (for initial experiments related to Figs. 1b and 2a).

Isolated NLuc-Hsp70 or NLuc-CD63 EV samples were incubated in PBS with or without 0.1% Triton X-100 and 50 μg/mL Proteinase K (AM2542, Ambion, Texas, USA) for 5 h at RT within 96-well plate. Then 20 μL of Nano-Glo^TM Live Cell assay reagent (N2011, Promega, Wisconsin, USA) was added on each sample to immediately measure the remaining NLuc activity in each well using iD3 SpectraMax microplate reader (Molecular Devices, California, USA). For Alix resistance assessment by immunoblot, Proteinase K was neutralized by adding 0.2 μL of boiled 0.2 μM PMSF immediately followed by loading buffer (100 °C, 15 min)¹⁴ .

Cells were incubated for 2 h in Alamar Blue HS (Invitrogen, Massachusetts, U.S.A.) and the fluorescence signal was measured according to the manufacturer’s instructions using the iD3 SpectraMax microplate reader (Molecular Devices, California, USA). For cell growth curves, cells were incubated with Alamar Blue HS at the indicated timepoints.

The following instruments were used: SpectraMax iD3 microplate reader, manufactured by MD (Molecular Devices, San Jose, USA); Costar microplate, manufactured by Corning (New York, NY, USA); Keithley 2602B digital source meter, manufactured by Keithley (Beaverton, OR, USA) in the United States; BX53M metallurgical microscope, manufactured by Olympus (Tokyo, Japan) in Japan; MPP i5000 multi-function wire bonder, manufactured by K&S(Haifa, Israel).

Supernatants from cultures infected with IBDV were collected and subjected to low-speed centrifugation (1,500×g for 5 min) to remove cell debris. The clarified cell supernatants were used to determine the extracellular virus titers by plaque assay using semisolid agar overlays followed by immunostaining as previously described (Méndez et al., 2015 (link)). GFP expression levels were used as readout of NDV-GFP and VSV-GFP virus replication. For this, cells were collected at 24 h pi, subjected to low-speed centrifugation, and cell pellets were resuspended in PBS. Cell samples were loaded in a 96-well black polystyrene microplate (flat bottom clear; Costar) and GFP was determined in an automate Spectramax iD3 microplate reader (Molecular Devices).