Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, then permeabilized, blocked with 0.5% bovine serum albumin, and incubated with primary antibodies (each 1:500 dilution) for 1 h at room temperature, followed by incubation with Cy3- or Alexa Fluor 488-conjugated secondary antibody for 1 h. DNA was counterstained with 0.5 μg/mL DAPI.
For high-content imaging analysis, images were obtained and analyzed using a CellInsight cellomics platform with HCS studio cell analysis software (Thermo Fisher Scientific). The Spot Detector BioApplication was used to quantify the total fluorescence intensity of mitochondrial signals and to count the number of nucleoli in each cell. Each cell was defined by a DAPI channel.
RNAi screening was described previously [12 (link)]. Briefly, 2,500 IMR-90 cells were seeded with 5 nM each siRNA in 96-well plates. After 3 days, cells were fixed and subjected to immunofluorescence using an anti-mitochondrial antibody (ab3298, Abcam). Sixteen images per well were taken using CellInsight with 20× magnification. The total area of mitochondria/cell was calculated using the Spot Detector BioApplication. Screens were performed in three biological replicates and hits were defined by the magnitude of change in the mitochondrial area, with statistical analysis using Student’s t-test between control siRNA and samples.
For high-content imaging analysis, images were obtained and analyzed using a CellInsight cellomics platform with HCS studio cell analysis software (Thermo Fisher Scientific). The Spot Detector BioApplication was used to quantify the total fluorescence intensity of mitochondrial signals and to count the number of nucleoli in each cell. Each cell was defined by a DAPI channel.
RNAi screening was described previously [12 (link)]. Briefly, 2,500 IMR-90 cells were seeded with 5 nM each siRNA in 96-well plates. After 3 days, cells were fixed and subjected to immunofluorescence using an anti-mitochondrial antibody (ab3298, Abcam). Sixteen images per well were taken using CellInsight with 20× magnification. The total area of mitochondria/cell was calculated using the Spot Detector BioApplication. Screens were performed in three biological replicates and hits were defined by the magnitude of change in the mitochondrial area, with statistical analysis using Student’s t-test between control siRNA and samples.
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