A total of 10,000 astrocytes were plated per well in 96-well black wall clear bottom plates (Greiner, Bedford, MA) and labeled with LysoTracker Red (Invitrogen) according to the manufacturer’s specifications and 20 ng/mL of Hoechst. Labeled live cells were imaged as previously described.6 (link)
96 well black wall clear bottom plates
Manufactured by Greiner
The 96-well black wall clear bottom plates are a type of laboratory equipment used for various assays and experiments. These plates feature a black wall structure and a clear bottom, which allows for optical detection and analysis of samples. The core function of these plates is to provide a standardized, multi-well format for conducting experiments in a controlled and efficient manner.
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2 protocols using 96 well black wall clear bottom plates
1
Astrocyte Labeling and Imaging
2
Lysosome Quantification in Cultured Neurons
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Neurons were plated 10,000 cells per well in 96-well black-wall clear-bottom plates (Greiner), labeled with LysoTracker® Red (Invitrogen) according to the manufacturer’s specifications, and 20 ng/ml of Hoechst. Labeled live cells were imaged at 10x magnification, six fields per well, in the DAPI and Cy3 channels using an IN Cell Analyzer 2200 (GE Healthcare). Images were analyzed with the IN Cell Workstation software (GE Healthcare) multi target analysis protocol. Briefly, nuclei were segmented by applying a Top Hat algorithm with a minimum area of 50 square μm and a sensitivity level of 50 to the DAPI channel. Lysosomes were defined as objects with a 1–3 μm diameter, segmented by 2 scales with a sensitivity level of 20 in the corresponding channel. Cell count, lysosome count, mean lysosome area and total lysosome area were calculated.
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