Transwell inserts with 8 μm wide pores were filled with a gel comprised of Collagen (Rat Tail Type I 1.6 mg/mL), Fibronectin (10μg/mL) and FCS (2%). LLC and CMT cells plated on top of each transwell were left to invade through the gel for 72h following a FCS concentration gradient. Transwells were fixed with 4%PFA and nuclei stained with DAPI. Confocal sections were taken every 1.5 μm in 5 independent fields per transwell with a 20x dry objective in a Nikon Eclipse Ti-E inverted microscope with A1R Si Confocal system using Nikon Software Elements. To quantify invasion levels, images acquired in the DAPI channel were imported on Fiji software. Cell count was obtained by thresholding for nuclear stain followed by automated counting. The percentage of total invading cells present at each depth in the collagen gel was determined. This was done by dividing the number of cells present in each layer by the sum of invading cells present in all layers.
A1r si confocal system
Manufactured by Nikon
Sourced in United States
The Nikon A1R Si Confocal system is a high-performance laser scanning confocal microscope designed for advanced imaging applications. It features a high-resolution silicon-based detector, enabling efficient light detection and high-quality imaging. The system is capable of capturing detailed, high-contrast images with minimal background noise.
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4 protocols using a1r si confocal system
1
Quantifying Cancer Cell Invasion
2
AuPAMAM/DNA Complexes Internalization Kinetics
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Cells were exposed to AuPAMAM/DNA complexes for 30 min, 1, 4, 24, or 48 h in 8-well chamber slides. After incubation, wells were aspirated and cells were rinsed with PBS. Lysotracker Yellow was diluted to 50 nM and 250 μl was added to the cells for 30 min. Following Lysotracker incubation, the cells were rinsed twice with PBS and complete media was added. Next, cells were fixed with 4% paraformaldehyde for 20 min. Cells were washed twice with 500 μl PBS, then one drop of NucBlue DAPI was added and incubated for 10 min. Finally, wells were aspirated and cells were resuspended in two drops of thawed ProLong Gold Mounting Media. Chamber slides were covered using coverslips and mounting media was left to cure overnight. Edges of the coverslip were sealed with nail polish. Confocal images were taken using a Nikon Ti-E widefield, inverted fluorescent microscope equipped with a Nikon A1-Rsi confocal system.
3
Bimolecular Fluorescence Complementation Assay
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For BiFC, single wells in the U96 Microwell plate were used to transfect protoplasts with plasmids encoding proteins of interest fused to the N-terminus of nEYFP and cEYFP. Empty vector (3 μg) encoding Enhanced Cyan Fluorescent Protein (ECFP) was added to each well as a transfection control. After transfection, protoplasts resuspended in 200 μl of W5 solution with 5 mM glucose [33 (link)] were transferred to black 96-well black glass-bottom plates (SensoPlate, Greiner Bio-One, www.greinerbioone.com ), sealed with transparent sealing tape (Thermo Scientific Nunc, cat. no. 236366), and returned to the growth chamber for overnight incubation. After incubation, the sealing tape was removed and the plate was screened with a Nikon A1Rsi confocal system (www.nikon.com ). See Additional file 8 for original files.
4
Quantitative Colocalization Analysis of Mitochondrial Morphology
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Colocalization analysis of MMs was performed as previously described[64 , 65 ] with modifications. A single isolated colony was picked from 24‐h SDAE plates, diluted in liquid YPD, and grown at 30 °C for 24 h. Cells were then re‐diluted in fresh YPD medium and grown statically in Ibidi µ‐dishes (Ibidi GmbH, Munich, Germany) for 24 h at 30 °C. The cells were washed, and then YPD medium containing 8 µm MM 1 and 10 nm MitoTracker Green (Thermo Fisher Scientific, MA, USA) was added. After a 30‐min dark incubation at 30 °C, the solution was replaced with fresh medium containing 40 nM FM 4–64 (Thermo Fisher Scientific, MA, USA). Cells were immediately imaged in a Nikon A1‐RSI confocal system mounted on a Nikon Ti‐E widefield fluorescence microscope (Nikon Corporation, NY, USA). Cells were imaged directly on the Ibidi imaging dish using a 60 × water immersion objective (numerical aperture of 1.27, 0.17 mm working distance). Colocalization was calculated in the Fiji version of ImageJ using the Colocalization Threshold tool and the Coloc‐2 plugin.
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