Bioprinted ARSs were imaged with an epifluorescent microscope (Eclipse TiE, Nikon, Melville, NY, USA) and acquisition software (MetaMorph, Molecular Devices, San Jose, CA, USA). MATLAB and ImageJ (National Institutes of Health, Bethesda, MD, USA) were used for further analysis. Confocal images of ARSs were acquired in a cell chamber (Attofluor, A7816, Thermo Fisher Scientific, Waltham, MA, USA) using a laser scanning confocal microscope (LSM800, Zeiss, Pleasanton, CA, USA) and ZEN lite software (Zeiss). Selected confocal images were converted to binary edge maps and transformed in Hough space to determine bioprinting-induced fiber alignment in MATLAB [35 ].
Attofluor
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Attofluor is a compact, fluorescence imaging system designed for live-cell imaging applications. It provides high-sensitivity fluorescence detection and supports a range of sample sizes and experimental configurations.
Automatically generated - may contain errors
4 protocols using attofluor
1
Bioprinted Artificial Retinal Scaffolds Imaging
2
Intracellular NO Measurement in Cardiomyocytes
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Intracellular release of NO was measured by DAF-FM Diacetate (DA), a membrane permeable dye that reacts with NO to form a fluorescent benzotriazole. Cardiomyocytes were plated on laminin-coated cover slides and after 2 h loaded with 20 µM DAF-FM DA in the dark at 37 °C for 30 min. After washing, cover slides were placed into a watertight cell imaging chamber (Attofluor, Thermo Fischer) at room temperature in Buffer B. Cells were visualized through a motorized digital inverted fluorescent microscope (iMIC; FEI, Graefelfing, Germany) with an oil immersion objective (×40 oil 1.35 NA; Olympus, Tokyo, Japan). Cells were excited at 500 ± 10 nm and emission was measured at 530 ± 15 nm.
3
Live Imaging of S. pombe Cells
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S. pombe cultures were grown to mid-log phase at 30°C in Edinburgh minimal media (EMM) supplemented with leucine, uracil, and adenine. Cells were harvested and washed once in phosphate-buffered saline (PBS). Cells were then resuspended in PBS, and 10 μl was deposited on an EMM-agarose pad before being mounted on ozone-cleaned circular coverslips (Thorlabs, #1.5H, ∅25 mm) and placed in a metal cell chamber for imaging (Attofluor, ThermoFisher). For replicative stress experiments, MMS was added to cultures at a final concentration of 0.03% and incubated for 5 hr before being processed for imaging.
4
Imaging Cell Migration and Durotaxis
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Cells transfected with plasmids encoding pmAKAR3 were cultured overnight in serum-free DMEM + 40 ng/ml epidermal growth factor (EGF), trypsinized, soybean trypsin inhibitor added, pelleted, and resuspended in DMEM 1% BSA + 25 or 40 ng/ml EGF. These cells were plated on fibronectin-coated coverslips and incubated for ∼4 h before imaging at low density to induce migration. Cells plated on hydrogels for durotaxis were incubated overnight in complete media then rinsed twice in modified Ringer’s buffer without phosphate (10 mM HEPES; 10 mM glucose; 155 mM NaCl; 5 mM KCl; 2 mM CaCl2; 1 mM MgCl2). All cells were refed modified Ringer’s buffer supplemented with 25 or 40 ng/ml EGF for imaging. Coverslips were mounted in a chamber (Attofluor; ThermoFisher) before imaging. Culture temperature was maintained at 35–37°C with hot air (ASI 400 Air Stream; Nevtek). Cultures of hydrogels cast in imaging dishes were mounted, warmed, and imaged in a suitable temperature controller (Delta T4; Bioptechs).
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