Netwell inserts

Placental large and small EVs were collected from human term placentae, using a well-established explant culture model [48 (link)]. Briefly, the placental tissues were dissected into explants of 400 mg wet weight and washed thoroughly to remove the contaminating blood. Then, the explants were cultured in 12-well culture plates with Netwell inserts (400 µm, Corning, Glendale, AZ, USA) in Advanced DMEM/F12 medium (In Vitro Technologies, Auckland, NZ), complemented with 2% FBS and 1% P/S overnight. The cultured medium was collected and centrifuged sequentially at 4 °C as follows: 2000× g for five minutes to remove the macro-vesicles/syncytial nuclear aggregates (SNAs) and cellular debris, 20,000× g for 60 min to collect large EVs, and 100,000× g for 60 min to collect small EVs (Sorvall wX+ ultracentrifuge (Thermo Fisher, San Jose, CA, USA)). The EV pellets were resuspended in PBS and were stored at 4 °C for future use.
In some experiments, the human placental explants were cultured in the presence of CellTracker^TM Red CMTPX (1 µg/mL, Thermo Fisher, Eugene, OR, USA) to generate fluorescently labelled placental large and small EVs.

Human foetal skin was cut to 1 cm² pieces that were scratch-inoculated with a virus on a petri dish and incubated for 3 h at 37°C prior to lifting onto NetWell inserts (Corning cat. # 29442). Tissues were incubated at the liquid–air interphase with the epidermis exposed to air, replacing the media every 2 days, at 37°C in humidified 5% CO₂ until harvesting at 4 or 8 DPI [74 (link)]. The culture medium was DMEM containing 10% foetal bovine serum (heat inactivated) and penicillin/streptomycin and amphotericin.

All experimental procedures were performed with 6 days post-fertilization (dpf) zebrafish larvae of the AB strain. Exposures and treatments were conducted in 12-well plates with Netwell inserts (Corning Inc.) to allow for the efficient transfer of larvae from plate to plate. Each well contained approximately 4.0 mL of solution, and all experimental solutions were diluted in E3 medium. The exposure and treatment paradigm consisted of transferring zebrafish larvae (20 per well) from a plate containing E3 medium onto a plate containing the diluted OP compound. The larvae remained in the OP solution for the experimentally dictated time before being transferred to two successive wash plates containing fresh E3 medium for 5 min each. Larvae were then transferred to a plate containing the oxime solutions and treated for 20 min. A final 5-min wash was conducted before the larvae were either transferred to individual wells of a 96-well plate (lethality and behavioral experiments) or humanely euthanized and placed in 1.5 mL conical tubes (AChE activity determination). All exposure and treatment experiments were performed in triplicate. Collected samples were frozen and stored at −80 °C for later analysis.

Cells were washed 3× with HBSS and fixed with 4% PFA for 20 min at room temperature. Cells were washed with PBS and glycine before addition of blocking solution (0.02% saponin, 10% normal goat serum (NGS), 1% BSA) for 1 h. Slides were incubated with primary antibodies overnight at 4 °C, washed with PBS + 0.02% saponin, and incubated with secondary antibody for 1 h [25 (link)]. Slides were mounted with Vectashield + DAPI (Vector Laboratories).
For slice culture: slices were floated in HBSS+/+ in 6-well plates containing Netwell™ Inserts (Corning). Samples were fixed in 4% PFA and 0.1% Triton X-100 for 1 h, rinsed 3× in PBS, then treated for 10 min of 1.5 mg/ml glycine. After three washes in PBS, slices were blocked in PBS containing 5% NGS for 1 h at room temperature. Slices were labeled with primary antibody (diluted in blocking) overnight. The following day, slices were washed 3× in PBS and labeled with Alexa conjugated secondary (1:500) for 1 h. After 3 washes in PBS, slices were stained with filipin labeling solution for 2 h, washed 3× with PBS, and mounted in ProLong Gold (ThermoFisher) and imaged by confocal microscopy. Calbindin was used to outline Purkinje cells, and filipin intensity was calculated using ImageJ.

Male Sprague Dawley rats (2 months old) were killed by decapitation under 4% isoflurane anesthesia, and brains were rapidly removed, placed in ice-cold oxygenated (O₂/CO₂, 95%/5%) Krebs–HCO₃^– buffer (in mm: 124 NaCl, 4 KCl, 1.25 KH₂PO₄, 1.5 MgCl₂, 1.5 CaCl₂, 10 glucose, and 26 NaHCO₃, pH 7.4), and sliced at 4°C using a brain matrix (Zivic Instruments). VTA slices (500 µm thick) were dissected at 4°C in Krebs–HCO₃^– buffer; each slice was transferred into a 12-well plate with Corning Netwell inserts containing 2 ml of ice-cold Krebs–HCO₃^– buffer. The temperature was raised to 23°C, and, after 30 min, the medium was replaced by 2 ml of fresh buffer (23°C). Slices were incubated under constant oxygenation (O₂/CO₂, 95%/5%) at 30°C for 4 h in an Eppendorf Thermomixer (5′), and the medium was replaced by fresh buffer and incubated for 30 min before the addition of any ligand. After incubation, the solution was discarded, and slices were frozen on dry ice and stored at 80°C until ERK1/2 phosphorylation was determined.