Fluorescent latex FluoSpheres beads (Molecular Probes, Eugene, OR) with mean diameters of 20 nm (F8786), 100 nm (F8801), and 500 nm (F8812) were chosen as model particles for penetration analysis. Their physicochemical characteristics such as size and ζ-potential were measured by DLS using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Nanoparticles were added to two-week-old PANC-1 or PANC-1/3 T3 (grown at initial cell number of 120 or 120:12, respectively) spheroids at final concentrations of 20 μg mL−1 and incubated for 24 hours. Spheroids were then washed twice with PBS to remove non-bound nanoparticles, frozen in HistoPrep tissue embedding medium, and cut into 10 μm sections using the Leica CM1510 cryotome (Leica, Wetzlar, Germany). To stain the cell nuclei, spheroid sections were incubated in 1 μg mL−1 Hoechst 33342 (Tocris Bioscience, Bristol, UK) in water for 5 minutes and air-dried before imaging.
Fluospheres beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
FluoSpheres beads are fluorescent microspheres designed for a variety of applications. They are available in a range of sizes and fluorescent dye options. The beads are uniform in size and highly fluorescent, making them suitable for use in flow cytometry, fluorescence microscopy, and other fluorescence-based techniques.
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7 protocols using fluospheres beads
1
Nanoparticle Penetration in 3D Tumor Spheroids
2
Isolation and Opsonization of Photoreceptor Outer Segments
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POS were prepared from freshly slaughtered cow eyes following a protocol adapted from a previous study (Klettner et al., 2011 (link)). Briefly, 12 retinas needed 10 ml homogenizer buffer (34% sucrose, 65 mM NaCl, 2 mM MgCl2) and were vortexed for 2 min. The solution was then centrifuged 4 min at 3′800 rpm at room temperature, allowing cell bodies to be pelleted and POS to remain in solution due to the osmolarity of the buffer. The supernatant was diluted in 2 volumes of HEPES 10 mM, gently mixed and centrifuged again. The supernatant was discarded and the pellet, constituted of POS, was resuspended in 5 ml of 10 mM HEPES and homogenized 30 times through a 23 gauge canula. The protein concentration was assessed by the Micro BCA protein stain assay (Perbio Science Switzerland SA, 23235).
Yellow-green fluorescent FluoSpheres® beads (Molecular Probes, F-8853) of 2.0 μm diameter were opsonized by POS. FluoSpheres were prepared with a ratio of 0.5 mg of POS per 43.3 million beads (10 μl of commercial solution), mixed together for 1 h on a rotary wheel at 4°C in the dark. Coated beads were separated by centrifugation at 6,600 rpm for 20 min at 4°C. Then the pellet was washed twice, using sterile 0.9% NaCl solution, and resuspended in 100 μl of this latter solution. POS-coated Fluospheres were used for phagocytosis assay just after preparation.
Yellow-green fluorescent FluoSpheres® beads (Molecular Probes, F-8853) of 2.0 μm diameter were opsonized by POS. FluoSpheres were prepared with a ratio of 0.5 mg of POS per 43.3 million beads (10 μl of commercial solution), mixed together for 1 h on a rotary wheel at 4°C in the dark. Coated beads were separated by centrifugation at 6,600 rpm for 20 min at 4°C. Then the pellet was washed twice, using sterile 0.9% NaCl solution, and resuspended in 100 μl of this latter solution. POS-coated Fluospheres were used for phagocytosis assay just after preparation.
3
GFP-expressing E. coli Bacteria Injection
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To obtain bacteria expressing GFP, E. coli bacteria (Sure 2, Agilent Technologies) were transformed with pFPV25.1 (Valdivia and Falkow, 1996 (link)) (Addgene plasmid # 20668) and grown overnight in a shaking incubator at 37 °C in Luria-Bertani's rich nutrient medium (LB broth) supplemented with ampicillin. After centrifugation, bacteria were resuspended in LB broth and cultures were normalized to OD600 = 0.5 using a spectrophotometer prior to injection. Yellow-green fluorescent FluoSpheres beads (1 μm diameter, Molecular Probes) were mixed with PBS to a final concentration of 0.08% solids per volume prior to injection. Cold-anesthetized 5-6 day-old females were injected into their thorax using a nanoinjector (Nanoject II, Drummond Scientific) with 69 nL of bacteria or 138 nL of fluorescent beads. Injected females were maintained with 10% (wt/vol) sucrose at 27 °C until hemocyte perfusions three days after bacteria injection or one day after beads injection.
4
Assessing Phagocytic Ability of Immune Cells
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Sorted slanDCs, CD1+ DCs, and CD14+ monocytes of healthy donors were maintained for 12 h in the presence or absence of 3 ng/ml or 30 ng/ml of FTY or FTYP in culture. To analyze, phagocytotic ability cells were treated with 1 μm carboxylate-modified yellow–green fluorescent FluoSpheres beads (Thermo Fisher Scientific, MA, USA) for 60 min at 37 °C. After cells were washed with PBS, incorporation of beads was evaluated by FACScan Calibur.
5
Fluorescent Bead Preparation for Imaging
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Commercially available FluoSpheres beads (carboxylate-modified orange fluospheres, 0.1 μm) from Thermo Fisher Scientific were used as test samples. A stock suspension containing 0.5 μL fluorescent beads in 25 μL of ethanol was prepared and then vortexed for 5 min. The stock solution was further diluted to 1:100 in Milli-Q water and 500 μL of the beads suspension was deposited on fluorodish glass dish (bottom poly-D-Lysine coated with a glass thickness of 0.17 mm) and left to air dry at room temperature. The glass bottom dish was thoroughly cleaned on the side of the objective immersion medium with ethanol prior to measurements.
6
Bronchial Epithelial Cell Imaging and Analysis
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Bronchial epithelia, differentiated on Snapwell permeable supports under ALC condition, were incubated for 3 hours with Coon’s modified Ham’s F12 medium (no serum) on the basolateral side. The medium was buffered with bicarbonate (2.68 g/L) or with 25 mM HEPES (pH = 7.2). After incubation, Snapwell supports were tilted by 10 degrees and 50 μL of PBS containing ATP 100 μM and yellow/green FluoSpheres beads of 200 nm diameter diluted 1:1000 (F8811, Life Technologies) were added to the apical side of epithelia just above the center of the permeable support. After 2 minutes, excess fluid was removed in a single step and snapwell supports were repositioned horizontally and transferred to a Nikon Eclipse TiE fluorescence microscope equipped with a 20x objective and GFP optical filter. An automated tile scan acquisition in the center of the permeable support (5 × 5 fields for a total surface of 5.8 mm2) was performed for each epithelium. The perfect focus system (PFS) was used to control the focal plane.
Image analysis was performed using Image J software implemented with a plug-in for texture analysis based on the Gray Level Co-occurrence Matrices method (GLCM texture). The GLCM for 0° and 90° and for a distance of 1 pixel were calculated and averaged. The following texture features were extracted: angular second moment, contrast, correlation, inverse difference moment and entropy.
Image analysis was performed using Image J software implemented with a plug-in for texture analysis based on the Gray Level Co-occurrence Matrices method (GLCM texture). The GLCM for 0° and 90° and for a distance of 1 pixel were calculated and averaged. The following texture features were extracted: angular second moment, contrast, correlation, inverse difference moment and entropy.
7
Fluorescent Polystyrene Bead Characterization
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A 100 nm-and 200 nm-sized, yellow-green fluorescent (505/515) polystyrene FluoSpheres beads were purchased from Life Technologies (Bleiswijk, The Netherlands). Bead concentrations were calculated based on the manufacturer's specifications. As shown in our previous work, these calculated concentrations correspond with the concentrations as measured by the high-resolution flow cytometry method (10) .
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