Cellvoyager cv1000 confocal scanner system

RAW 264.7 cells between passages one and six were grown in complete DMEM to a confluency of 70–90%. Cells were scraped, washed once, resuspended, and diluted to 5x10⁵ cells/mL. Cells (100 μL) were added to a black 96-well glass-bottomed plate (Brooks Life Sciences) and incubated for 23.5 hours at 37°C with 5% CO₂. At 23.5 hours the medium was exchanged for 100 μL of 3.4 μM Nile red in FluoroBrite DMEM and incubated for 30 minutes. The medium was exchanged for 150 μL of 0.314 μM Nile red in FluoroBrite DMEM. Compounds were added in 50 μL to obtain the desired concentration. The final volume per well was 200 μL, with 0.235 μM of Nile red and 0.32% DMSO. Cells were time-lapse imaged on a Yokogawa CellVoyager CV1000 Confocal Scanner System with a 40x/0.6NA objective (ex561/em617/73 nm) in an environmentally controlled chamber every hour for 17 hours. Two fields per well were imaged with each field comprised of five images sampled over a z-dimension of 15 μm. The resulting images were converted into maximum intensity projections and used to evaluate morphological changes in the cell membrane over time.

Experiments were performed with RAW 264.7 cells between passages one and six. Cells were grown in complete DMEM to a confluency of 70–90%. Cells were scraped, washed once, resuspended and diluted to a final concentration of 5x10⁵ cells/mL. Cells (100 μL) were transferred to a 96-well black glass-bottomed plate (Brooks Life Sciences) and incubated for 23.5 hours at 37°C with 5% CO₂. At 23.5 hours, the medium was exchanged for a 100 μL of FluoroBrite DMEM containing 100 nM of TMRM. Thirty minutes later, the medium was exchanged for 150 μL of FluoroBrite DMEM. Cells were time-lapse imaged on a Yokogawa CellVoyager CV1000 Confocal Scanner System with a 20x/0.75NA objective and an environmentally controlled chamber for 30 minutes with images acquired every 10 minutes. Compounds were then added to the plate with a multichannel pipet as 50 μL aliquots to obtain the desired concentration and a final volume of 200 μL. All wells contained a final concentration of 0.4% DMSO except for the CCCP control, which required 0.5% DMSO to remain in solution. Cells were imaged every 10 minutes for 80 minutes. Two fields per well were imaged with each field comprising five images sampled over a z-dimension of 15 μm. Images were converted into maximum intensity projections and the TMRM foreground signal was extracted via a MATLAB R2018a (MathWorks) script and normalized to time zero for each field.

Experiments were performed with RAW 264.7 cells between passages one and six. Cells were grown in complete DMEM to a confluency of 70–90%. Cells were scraped, washed, resuspended and diluted in complete DMEM to a final concentration of 5x10⁵ cells/mL. Cells (100 uL) were transferred to a 96-well glass bottom plate (0.17mm, Brooks Life Sciences) and incubated for 23.5 hours at 37° C with 5% CO₂. The medium was exchanged for 100 uL of complete FluoroBrite DMEM with TMRM [100 nM] and incubated for 30 minutes. The medium was exchanged for 150 uL of complete FluoroBrite DMEM. Cells were imaged on a Yokogawa CellVoyager CV1000 Confocal Scanner System with a 20x/0.75NA objective and an environmentally controlled multi-well chamber over 30 minutes with images acquired every 10 minutes. Compounds were added (50 uL) with a multichannel pipet to obtain the desired concentration and a final volume of 200 uL with 0.5% DMSO. Cells were imaged over 16 hours with acquisition every 30 minutes of two fields of view per well. Five images over a z-dimension of 15 μM were sampled per field. The resulting volumes were converted into maximum intensity projections and TMRM foreground signal was extracted via a MATLAB R2018a (MathWorks) script and normalized to time zero for each field.