Opera phenix

SH-SY5Y cells were seeded in 384-well plates at 8 × 10² cells/well, incubated for 15 h, and then replaced with DMEM/F12 medium containing 7.5 µM retinoic acid, 1% FBS and 1% penicillin-streptomycin for 24 h. The medium was further changed twice, every 24 h. The medium was then replaced with the compound-containing medium and incubated for 48 h. After removing medium, the cells were fixed by adding 4% PFA for 15 min. Each well was washed with PBS, added 3% BSA, and incubated for 60 min at 37 °C. βIII Tubulin antibody as primary antibody (MAB1195; R&D Systems, Minneapolis, MN) and Alexa Fluor 488 goat anti-mouse IgG as secondary antibody (A-11001; Thermo Fisher Scientific) were used to stain the cells. Cell axon length was measured using Opera Phenix (PerkinElmer).
Primary cerebellum neurons were seeded in 96 well plates at 1 × 10⁴ cells/well, cultured for 24 h. The medium was then replaced with the compound-containing medium and incubated for 48 h. After removing medium, the cells were fixed by adding 4% PFA for 15 min. Each well was washed with PBS, added 3% BSA, and incubated for 60 min at 37 °C. Tau1 antibody as primary antibody (MAB3420A4; Merck/Sigma-Aldrich, Munich, Germany) and Alexa Fluor 488 goat anti-mouse IgG as secondary antibody (A-11001; Thermo Fisher Scientific) were used to stain the cells. Cell axon length was measured using Opera Phenix (PerkinElmer).

CFBE41o^– cells stably expressing F508del-CFTR and the HS-YFP
were plated at a low density (5000 cell/well) on 96-well plates suitable
for high-content imaging. After 6 h, cells were treated with different
concentrations of test compounds or vehicle alone (DMSO). Cell proliferation
was monitored (by exploiting the YFP signal to determine the area
covered by cells) for 48 h using the Opera Phenix (PerkinElmer) high-content
screening system. Alternatively, to monitor the cytotoxic effect of
high concentrations of test compounds, CFBE41o^– cells
were plated (10,000 cell/well) and treated (after 6 h) with test compounds
or vehicle alone (DMSO). After 24 h, cells were counterstained with
Hoechst 33342 and propidium iodide to visualize total cells and apoptotic
cells, respectively, and imaged by using the Opera Phenix (PerkinElmer)
high-content screening system. Data are expressed as means ±
SEM, n = 6. Reproducibility of results was confirmed
by performing three independent experiments. Statistical significance
was tested by parametric ANOVA followed by the Dunnet multiple comparisons
test.

The different microneedles replicated from silicone molds were observed under a stereomicroscope (JSZ6S, Jiangnan Novel Optics) and captured by CCD (Oplenic digital camera). The integrity of the microneedles was recorded by immersing them in phosphate-buffered saline (PBS, pH 7.4) after days 1, 3, and 7. Surfactin extraction from B. subtilis was evaluated by a liquid phase mass spectrometer (LCMS6120, Agilent Ltd.). Living bacteria in the LMNs were stained by SYTO and observed by Opera Phenix (PerkinElmer Inc., UK).