Rubber tipped cell scraper

TC is a photosensitizer and is known to generate ROS upon irradiation by UV light or Cerenkov radiation 69 (link), 70 (link). ROS measurements were carried out at RT using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, Thermo Fisher Scientific, Waltham, MA) as ROS indicator. RaST treatments were performed as described in the in vitro RaST section. After the VLA-4-TC-NM RaST and the controls were added to the appropriate wells, H₂DCFDA was added to each well as follows. H₂DCFDA was reconstituted in DMSO immediately prior to being added to each well at 5 µM final concentration and the plates were incubated in humidified atmosphere at 37 °C/5% CO₂ for 72 h. At the end of the incubation period, the dye was excited at 495 nm and the emitted fluorescence was detected at 520 nm with a Synergy/NEO2 multi-mode reader (BioTek, Winooski, VT). The cells were mechanically harvested with a rubber-tipped cell scraper (Sarstedt, Newton, NC), washed twice with PBS, lysed with 200 µL RIPA buffer at 4 °C for 30 min and the total protein content was determined with the BCA assay. The data were reported as fluorescence intensity per µg of protein.

The cells were grown in MEBM supplemented with MEGM Single Quots and cholera toxin (Lonza, Basel, Switzerland) on cover glasses placed inside petri dishes (Thermo Fisher Scientific, Nunc^™, 8.8 cm²). After reaching ∼70% confluence, equivalent to ~2 million cells, the cells were exposed to one of the 27 compounds listed in Table 1 for 24 h. Three biological replicates were created in order to catch biological variation. After 24 h incubation in presence of test agents, cells were harvested. Cover glasses were carefully lifted from the bottom of the petri plates using tweezers and then dipped into the stirring water of the washing unit to rinse both sides of the cover glasses. They were then quickly placed in the quenching solution (−20 °C 80% methanol) followed by detachment of cells using a rubber-tipped cell scraper (Sarstedt, Nümbrecht, Germany). Detached cells were transferred to 1.5 ml conical centrifuge tubes and centrifuged for 10 min at 21,000×g 4 °C. The supernatants collected were freeze dried using centrifugal vacuum concentrator (1–2 h).
The whole sample preparation procedure was split into four weeks, creating four sample batches. Each batch contained three Mebendazole replicates (to catch batch effects), three control samples (with three replicates each) being cells treated with only 0.01% DMSO and six blank samples, containing no cells or drugs, only DMSO.