Opera phenix high content microscope

IL-6 protein was measured in the supernatant of astrocyte-conditioned medium. Undiluted supernatant was collected following the indicated culture times and analyzed on a PHERAstar plate reader (BMG Labtech) using an AlphaLISA IL-6 kit (Perkin Elmer), per the manufacturer’s instructions. Concentration of IL-6 was calculated based on the standard curve provided in the AlphaLISA kit. Following collection of the supernatant, cells were live imaged with Calcein Green AM and Hoechst to quantify cell number. PBS containing calcium and magnesium supplemented with Hoechst 33342 (1:1000, Thermo-Fisher Scientific) and Calcein Green AM (50 μg vial resuspended in 50 μL DMSO, 1:1000, Thermo-Fisher Scientific) was applied to the cultures for 10 minutes at 37C. Cells were washed 3X with PBS and the entire well was imaged using a 5X objective on a Perkin Elmer Opera Phenix high content microscope. The number of Calcein Green AM positive nuclei were quantified using Harmony software and used to normalize protein expression values to determine IL-6 concentration on a per-viable cell basis.

Following clearing, 384-well plates with spheroids were imaged on an Opera Phenix high-content microscope (PerkinElmer) using a 20x water objective (NA 1.0, HH14000421, PerkinElmer), with 2x digital camera pixel binning, which yields a xy pixel size of 640 nm. Image stacks were taken with 5 µm step sizes to a total depth of 200 µm for U87 spheroids and 300 µm for T47D spheroids. Excitation/emission wavelengths used were 405/435-480 for Hoechst, 488/500-550 for GFP, 561/570-630 for AlexaFluor 568, and 640/650-760 for CT Deep Red.
For time course and seeding density experiments, experiments were repeated in triplicate, with 28 T47D wells imaged per condition per experiment. Fifty-six U87 wells were imaged per condition per experiment, except experiment 1 1000 cell condition, and experiments 2 and 3 250 cell condition, for which 28 wells were imaged. For CT experiments, experiments were repeated in duplicate, with 2 plates per independent experiment. Fourteen wells were imaged for the 100% CT condition per plate, and 28 wells were imaged per plate for 50%, 25%, 12.5%, 6.25%, 3.125%, and 0% conditions. The PH3 data shown is representative of several repeated experiments. Sixteen wells were imaged per treatment condition, 14 wells for the no primary control, and 28 wells for the untreated condition.

Rat cortical neurons were prepared by a Neuronal Cell Culture unit of University of Helsinki from late embryonic stage (E17–18) rat embryos. Animal work was performed in accordance with the ethical guidelines of the European Convention and regulations of the Ethics Committee for Animal Research of University of Helsinki. Neurons were plated in a 96-well white glass-bottom plate (Corning) at a plating density of 20,000 cells per well, grown for 7 days in vitro, and transduced with an AAV9 encoding mCherry-Dr-TrkA under CAMKII promoter for 24 h. After that, the transduction medium was changed to a medium with 25 μM of BV. For detection of apoptosis, plates with neurons were kept under either 660 nm or 780 nm light for 6 h. As a control for apoptosis, neurons were treated with 100 nM staurosporine. Imaging of neurons was performed using an Opera Phenix High-Content microscope (Perkin Elmer) equipped with a 60× water-immersion objective.