After Aβ1–42 intoxication (Supplementary Table 1 ), cells were fixed, permeabilised, and non-specific sites were blocked with a solution of phosphate buffered saline (PBS; PanBiotech) containing 0.1% of saponin (Sigma) and 1% FCS. Then, cells were incubated with primary antibody, co-stained with the other primary antibody, and revealed with secondary antibodies (Supplementary Table 3 ). Nuclei were counter-stained with Hoechst (Sigma). 10 to 40 pictures per well were taken in each experiment using the InCell Analyzer™ 1000 (GE Healthcare, France). Analysis was done using Developer software (GE Healthcare) assessing the overlap between MAP2 on one side, and MetO, Cyto c, Caspase 3 or pTau staining on the other side. PSD95 and Synaptophysin total surface overlap was quantified (in μm2) and computed for each condition. Results were expressed as the number of overlapping stained cells per field and reported as a percentage of vehicle control.
Developer software
Manufactured by GE Healthcare
The Developer software is a laboratory equipment product from GE Healthcare. It is a software application that assists in the analysis and processing of medical images and data. The core function of the Developer software is to provide tools and features for enhancing, manipulating, and interpreting medical imaging data to support healthcare professionals in their diagnostic and treatment processes.
Automatically generated - may contain errors
Lab products found in correlation
In cell analyzer 1000, by GE Healthcare (1 mentions)
Hoechst, by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by PAN Biotech (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
In cell analyzer 6000, by GE Healthcare (1 mentions)
3 protocols using developer software
1
Immunofluorescence analysis of Aβ-induced neurodegeneration
2
Immunofluorescence Microscopy Analysis
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Cells were seeded on coverslips in 24-well plates (10,000–15,000 cells per well) for observation with confocal microscopy. The cells were treated with compounds the next day. Cells were fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.5) with 0.1% Triton X-100 for 15 min at room temperature. Samples were permeabilized with 0.3% Triton X-100 in PBS and blocked in 3% bovine serum albumin (BSA), 0.1% Tween 20 in PBS. Primary and secondary Alexa-Fluor-conjugated antibodies were diluted in 3% BSA, 0.1% Tween 20 in PBS. DNA was counterstained with DAPI (Invitrogen). Images were acquired in a Zeiss LSM-510 confocal microscope with the 488 oil objective. For high-throughput immunofluorescence, cells were seeded in 96-well plates (2500–3000 cells per well). The cells were treated with compounds the next day. Cells were fixed and stained as described above. DNA was counterstained with DAPI. Quantification was done by automatic acquisition of images (500 cells per well) using the In Cell Analyzer 2000 (GE Healthcare) and Developer software (GE Healthcare).
3
Cell Proliferation Assay Protocol
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The proliferation assay has been described elsewhere [20 (link)]. The cells were plated into 24-well plates at 1 × 103 cells/well. The next day, the cells were treated with the indicated reagents. Images of the cells in randomly selected wells, with nine fields per well, were captured, and continued to be obtained in the same fields at 24-h intervals using IN Cell Analyzer 6000 (GE). The images were deconvoluted using Developer software (GE) to recognize the cells morphologically, and the cells were counted.
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