Automated quantification of F-actin fibers was performed using a custom pipeline in Cell Profiler (Stirling et al., 2021 ). Briefly, raw micrographs of phalloidin staining were exported from Volocity 6.1.2 software (Perkin Elmer) and imported into Cell Profiler. Images were uniformly enhanced for contrast, and primary objects were identified. Additional filtering was applied to ensure that detected objects corresponded accurately to F-actin staining and were visualized by pseudo-colored images where different colors identify different fibers (shown in figure panels). Detected objects were automatically quantified, and data were exported from Cell Profiler and plotted in Prism 9 software (GraphPad Software), with each data point representing the average quantification of F-actin filaments from all acquired images (10–15) in each treatment group from a single independent experiment.
Cell profiler
Manufactured by PerkinElmer
CellProfiler is a free, open-source software application designed for quantitative analysis of biological images. It enables users to build complex image analysis pipelines without requiring advanced programming skills.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using cell profiler
1
Automated F-Actin Fiber Quantification
2
Immunofluorescence Staining Protocol
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Cultures were fixed with 2% PFA for 20 min at room temperature (RT), rinsed with PBS, and antigen retrieved in citrate buffer pH 6.0 (ThermoFisher) in water bath at 80 °C for 5 min. Samples were left to rest for 10 mins at room temperature before being permeabilised and blocked in PBS, 10% donkey serum and 0.01% of Triton X-100 for 10 min. Incubation of primary antibody in PBS and 10% donkey serum was performed overnight at 4 °C. Samples were washed with PBS and subsequently incubated in species-appropriate Alexa Fluor© secondary antibody in PBS with 10% donkey serum for 1 h at RT. The list of primary and secondary antibodies used for immunostaining is available on Zenodo (10.5281/zenodo.10650790). Images were acquired on the Opera Phenix High-content Screening system (PerkinElmer) or the Invitrogen EVOS ™ FL Auto (ThermoFisher) cell imaging system and subsequently processed on Harmony (Perkin Elmer; RRID:SCR_018809), ImageJ (RRID:SCR_003070) or CellProfiler (RRID:SCR_007358), respectively.
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