MDCK cells were seeded in flat-bottom 12-well plates at a density of 1×106 cells per well. When the cells reached confluency, 20 μg mL−1 agSWCNHs and SWCNHox in complete medium were added and incubated with the cells for 24 h at 37°C to allow endocytosis. After incubation, single-cell suspension was obtained and dropped onto glass bottom culture dishes. The internalization of agSWCNHs and SWCNHox was monitored by a confocal laser scanning microscope (Leica TCS SP8) at 561 nm, the wavelength at which the nanocarbon showed a strong reflect light. The images from agSWCNHs or SWCNHox and bright field were merged using the image acquisition software of Leica. The mean intracellular fluorescence intensity or reflected light intensity was analyzed with Leica Qwin software.
Image acquisition software
Manufactured by Leica
The Image acquisition software by Leica is a tool designed to capture high-quality digital images from various imaging devices. It provides a user-friendly interface for controlling and managing the image acquisition process.
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3 protocols using image acquisition software
1
SWCNH Internalization in MDCK Cells
2
Quantitative Analysis of Neuronal Synaptic Puncta
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Images were acquired using a 63× oil objective. Settings were chosen to exclude signal saturation in each channel using Quick Lookup Tables (QLUT) available in the Leica image acquisition software. The maximum intensity projection of each image was then analyzed using Puncta Analyzer, an ImageJ plugin written by Barry Wark and obtained upon request from c.eroglu@cellbio.duke.edu (Ippolito and Eroglu, 2010 (link)). The fibers of the neurons extensively arborize across the dish although the smaller fibers are not captured in our images due to oversaturation of the proximal dendrites and soma. Counting puncta in these proximal dendrites and soma allows us to ensure that we are counting co-localized sites associated with a single neuron. We confirmed neuron-specific staining by demonstrating that, upon overexposure to MAP2, Shank and VAChT puncta are confined to the MAP2-positive fibers. The number and size of synaptic puncta on neuronal cell bodies and proximal dendrites (<50 μm from the soma) were quantified using identical threshold values for all cells in both conditions. The number of synaptic puncta was normalized to the MAP2-positive area. The size of synaptic puncta, defined as the total area of co-localized pre- and post-synaptic markers, was calculated using Puncta Analyzer.
3
Internalization of F-B-SWCNHox in MDCK Cells
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MDCK cells were seeded in flat-bottom 12-well plates at a density of 1×106 cells per well. When the cells reached con-fluency, 20 μg mL−1 F-B-SWCNHox in complete medium were added and incubated with the cells for 24 h at 37°C to allow endocytosis. To analyze the state of the probe and to assess the reliability of the method, both F-B-SWCNHox centrifugal supernatant (20,664 ×g 30 min) and the cells without any treatment were used as control. After incubation, single-cell suspension was obtained and dropped onto glass bottom culture dishes. The internalization of F-B-SWCNHox was monitored under a bright-field confocal microscope at 488-nm excitation wavelength for detecting FITC. The images from bright-field and FITC channel were merged using the image acquisition software of Leica. The mean intracellular fluorescence intensity was analyzed with Leica Qwin software.
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