Autophagy quantification was performed as described in Jacomin et al. [26 (link)]. Briefly, HeLa GFP-LC3 cells were seeded on 96-well plate (#655090, Greiner) at 7500 cells per well; 24 h later, cells were treated with DMSO at 0.25% and rapamycin at 250 mM, as bioinactive and bioactive controls, respectively, or with the extracts at the indicated dilution, for 2 h. After fixation (4% paraformaldehyde), the DNA was stained with 1 µg/mL Hoechst. Image acquisitions were performed on an ArrayScanVTI using a Zeiss 20× Plan-Neoflur air objective (10 fields per well). Images were automatically analyzed with SpotDetector Bio-Application of Thermo Scientific (Illkirch, France) HCS Studio v6.5.0, allowing us to count and extract the GFP-LC3 dots per cell parameter. Results are presented as a proportion of the cells containing less than three autophagosomes (ATG), between three and eight, and more than eight ATG.
Spotdetector bioapplication
Manufactured by Thermo Fisher Scientific
Sourced in France
The SpotDetector BioApplication is a software tool designed for the analysis and quantification of protein spot patterns in 2D gel electrophoresis experiments. It provides automated spot detection, background subtraction, and spot volume calculation to assist in the comparative analysis of protein expression levels across multiple samples.
Automatically generated - may contain errors
Lab products found in correlation
Arrayscan vti hcs reader, by Thermo Fisher Scientific (1 mentions)
Arrayscan vti, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Plan neofluar 0.4 na, by Zeiss (1 mentions)
Arrayscan xti hca reader, by Thermo Fisher Scientific (1 mentions)
Tcs sp2 confocal fluorescence microscope, by Leica (1 mentions)
Cellinsight cx7, by Thermo Fisher Scientific (1 mentions)
Fluoroshield, by Merck Group (1 mentions)
7 protocols using spotdetector bioapplication
1
Quantification of Autophagy Levels
2
Quantitative Analysis of Fluorescent Transgenes
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Quantitative analysis of animals expressing fluorescent transgenes was performed using the ArrayScan VTI HCS Reader (Cellomics, ThermoFisher, Pittsburgh, PA, USA) fitted with a 2.5x objective and a 0.63x coupler as previously described [11] . Briefly, 35 L4 stage animals were placed into wells of 384-well plates containing DMSO or Flu to a final concentration of 50 µM. Worms were sorted using the COPAS Biosort to ensure that only animals of the similar age and fluorescence intensities were used for the experiment. Following incubation at 20°C for 24 hours, sodium azide was added to a final concentration of 50 µM to anesthetize the animals. Animals were then imaged on the ArrayScan VTI (Thermo Scientific) and protein aggregates were quantified using the SpotDetector BioApplication (Thermo Scientific).
3
Quantitative Analysis of Autophagy Flux
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Image analysis of H4-GFP-LC3 cells fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with 3 µg/ml DAPI (Sigma-Aldrich), using ArrayScan HCS 4.0 Reader with a 20× objective lens (Cellomics ArrayScan VTI; Life Technologies). The Spot Detector BioApplication (Thermo Fisher Scientific) was used to acquire and analyze the images after optimization. Images of 1,000 cells for each compound treatment were analyzed to obtain the mean cell number per field, fluorescence spot number, area, and intensity per cell. DMSO and rapamycin were used as negative or positive controls, respectively. The percentages of changes of LC3-GFP were calculated by dividing with that of DMSO-treated samples. The images were also analyzed by using a conventional fluorescence microscope for visual inspection. The experiments were repeated three times with consistent results.
4
Calcium Imaging of Primary Cortical Neurons
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Primary cortical neurons were loaded with 2 µM Fluo‐4 (Invitrogen) in KRH (Krebs’–Ringer’s–HEPES containing (in mM): 125 NaCl; 5 KCl; 1.2 MgSO4; 1.2 KH2PO4; 25 HEPES; 6 glucose; 2 CaCl2; pH 7.4) for 30 min at 37°C and then washed once with the same solution. Stimulation was performed automatically by using the liquid handling system of the ArrayScan XTI HCA Reader (Thermo Fisher Scientific). To stimulate, one dose of NMDA (100 µM at the rate of 50 µl/s) was added while images were digitally acquired with a high‐resolution camera (Photometrics) through a 20× objective (Zeiss; Plan‐NEOFLUAR 0.4 NA). Hoechst fluorescence was imaged as well. 70 frames were acquired at 1 Hz with 40 ms exposure time for Fluo‐4 and 25 ms exposure time for Hoechst. At least 8 baseline images were acquired before stimulation. The analysis was done with HCS Studio software using SpotDetector bioapplication (Thermo Fisher Scientific). Hoechst‐positive nuclei were identified and counted, and the mean intensity of the Fluo‐4 signal was measured in the cell body area of each cell; background intensity was measured and subtracted from the mean intensity. Only cells with neuronal morphology were included in the analysis. Calcium responses were measured as ∆F/F0.
5
Quantifying Autophagy Flux by LC3 Puncta
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Cells were fixed with 4% paraformaldehyde for 30 min, permeabilised with 0.2% Triton X-100 (Sigma-Aldrich, T8787) for 10 min, and blocked in PBS containing 5% bovine serum albumin (BSA; Beyotime, ST023) for 30 min. Cells were incubated with anti-LC3 (1:100) at 37°C for 1 h, followed by secondary antibodies conjugated to Alexa Fluor 488. The fluorescence signals were visualized with a TCS SP2 confocal fluorescence microscope (Leica). The Spot Detector BioApplication (Thermo Fisher Scientific) was used to acquire and analyze the images after optimization. Images of 500–1,000 cells for each treatment group were analyzed to obtain the mean LC3 fluorescence punctae number per cell (Tang et al., 2010 (link)). (n = 5 each group).
6
Lysosomal Damage Assay Using U2OS Cells
7
Immunocytochemistry of KC Precursors
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Cultures of KC precursors at 50% confluence performed in 96-well plates coated with type I collagen (Biocoat, BD) were fixed with 4% paraformaldehyde for 10 minutes at 4 °C and permeabilized with PBS-Triton 0.1%. Staining was performed using nonconjugated primary antibodies, revealed using a fluorochrome-conjugated secondary antibody. Negative controls corresponded to the staining procedure without primary antibody and showed no signal (antibodies are listed in Supplementary Table S7). Nuclei were counterstained with DAPI (Fluoroshield, Sigma-Aldrich). Image acquisition and analysis were performed using a high-content imaging platform CellInsight CX7 (Thermo Fisher Scientific, Waltham, MA) equipped with the SpotDetector BioApplication.
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