Spectrolinker xl 1000 uv crosslinker

To ensure the impermeability of the printed NOCs, they were covered with silicone (SYLGARD™ 184 Silicone Elastomer Base and Curing Agent; Fa. Dow Europe GmbH, Midland, MI, USA) by dip coating and drying overnight at room temperature. The bottom of the NOC was coated with grease (high vacuum grease; Dow Corning, Midland, MI, USA) to mount two circular glass coverslips on the bottom. A Ø9 mm glass coverslip for cell growth was placed below the canal followed by a Ø30 mm glass coverslip (both Menzel-Gläser, Braunschweig, Germany) to cover the whole NOC bottom (Figure 2B,C). The NOC was gently pushed against the glass coverslips to provide a liquid-tight seal mediated by the grease. The NOC, the glass coverslips, and the grease were decontaminated by UV radiation (Spectrolinker XL-1000 UV Crosslinker; Spectroline, Westbury, NY, USA) before use.
The inner glass bottom surface of the NOC (i.e., both compartments and the connecting canal) was coated with poly-DL-ornithine (0.1 mg/mL; Sigma Aldrich, Taufkirchen, Germany) and Natural Mouse Laminin (0.01 mg/mL; Invitrogen, Karlsruhe, Germany) to support cell adhesion and neuronal growth.

Parasites expressing the synthetase/tRNA cassette and mutant protein of interest constructs were allowed to infect HFFs overnight at a multiplicity of infection of approximately 3 and the growth medium was replaced with fresh medium supplemented with 1 mM Azi (Bachem, Torrance, CA). Following a 24-hour incubation period and lysis of the host cells, extracellular parasites were collected by centrifugation and resuspended in an adequate volume of PBS for UV irradiation (approximately 10⁷ parasites per mL of PBS, in tissue culture plates). The plates containing resuspended parasites were floated on an iced water bath and placed without lids in a Spectrolinker XL-1000 UV crosslinker (Spectroline, Westbury, NY) equipped with 365-nm (UV-A) bulbs. Parasites were irradiated for 20 minutes with periodic mixing using a micropipette. The cells were then collected by centrifugation and lysed for either co-IP or directly in sample buffer for SDS-PAGE.

To determine effects of UV-induced tissue damage on larval cold nociception and sensitization, larvae were irradiated by exposure to 10–14 mJ/cm² UV-C (as previously described [38 (link)]) and then allowed to recover on food in a 25°C incubator before being tested in the nociceptive assays 4, 8, 16 or 24 hours later. For this, 3^rd instar larvae of similar size were selected 4–5 days after egg laying and irradiated, then tested in the cold assay after recovery from UV. Before irradiation, larvae were immobilized on a cold slide for a few minutes, then fine-tipped forceps were used to position the larvae dorsal side up in a row along the length of the slide. The spectrolinker XL-1000 UV crosslinker (Spectroline) was warmed up, and the UV dose was measured with a hand-held UV spectrophotometer (AccuMAX XS-254, Spectroline) just prior to exposure to get an accurate reading. All UV doses fell within 10–14 mJ/cm², which have been shown to induce epidermal cell death [55 (link)] while sparing the peripheral sensory neurons from significant morphological changes [46 (link), 56 ]. Larvae were then administered UV (actual dose recorded), and carefully placed into a vial of food with a paintbrush to recover for a variable amount of time before being tested in nociceptive assays.