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Fluosphere

Manufactured by Thermo Fisher Scientific
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FluoSpheres are fluorescent microspheres designed for a variety of research and analytical applications. They are available in a range of sizes and colors, and can be used as calibration standards, flow cytometry controls, and fluorescence microscopy markers. The core function of FluoSpheres is to provide stable, uniform fluorescent particles for various experimental and measurement purposes.

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Lab products found in correlation

Fitc dextran, by Merck Group (5 mentions) Influx flow cytometer, by BD (5 mentions) Aptes, by Merck Group (4 mentions) Lsm 780, by Zeiss (4 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions) D glucose, by Merck Group (4 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Zetasizer nano z, by Malvern Panalytical (3 mentions) Hoechst 33258, by Merck Group (3 mentions) Fv1000, by Olympus (3 mentions) Accuri c6, by BD (3 mentions) Bind silane, by Merck Group (3 mentions) Sodium dodecyl sulfate (sds), by Merck Group (3 mentions) Facscalibur flow cytometer, by BD (3 mentions) Matlab, by MathWorks (3 mentions) Proparacaine, by Bausch & Lomb (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Ab6586, by Abcam (2 mentions) Cellquest pro, by BD (2 mentions)

283 protocols using fluosphere

1

In Vitro Phagocytosis Assay

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In vivo phagocytosis in differentiated MGL cells was detected using 1 μm polystyrene yellow-green FluoSpheres (Life Technologies, Carlsbad, CA, USA). A total of 5 μL of FluoSpheres was directly added to MGD media (107 beads/mL/105 MGL cells) and incubated for 24 h. The FluoSpheres engulfment and clearance around the MGL cells were visualized using an EVOS FL microscope (Thermo Fisher, Waltham, MA, USA).
Ihnatovych I., Birkaya B., Notari E, & Szigeti K. (2020). iPSC-Derived Microglia for Modeling Human-Specific DAMP and PAMP Responses in the Context of Alzheimer’s Disease. International Journal of Molecular Sciences, 21(24), 9668.
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2

Infarct Size Measurement in I/R Model

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Infarct size was measured 24 hours after I/R surgery as described previously.13 After thoracotomy, the LAD was religated, and 50 μL green fluorescent FluoSpheres (Molecular Probe, Carlsbad, CA) was injected into the LV of the heart to delineate the area at risk (FluoSpheres‐negative area). The heart was then excised, fixed, and sectioned into 1‐mm perpendicular sections. Sections were stained with 2,3,5‐triphenyltetrazolium chloride (TTC) (Sigma Chemical, St. Louis, MO) solution to determine the infarcted myocardium (TTC‐negative area). The infarction area (IA) and the area at risk (AAR) were determined for each slice using a computer planimetry and NIH Image software.
Bae S., Park M., Kang C., Dilmen S., Kang T.H., Kang D.G., Ke Q., Lee S.U., Lee D, & Kang P.M. (2016). Hydrogen Peroxide‐Responsive Nanoparticle Reduces Myocardial Ischemia/Reperfusion Injury. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 5(11), e003697.
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3

Elastic Substrates for Cell Migration

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We have used two types of elastic substrates. In the experiments using the MyoII mutant cell lines, the substrate used is a gelatin gel with yellow latex beads of 0.1 μm diameter (FluoSpheres; Molecular Probes, Eugene, OR) embedded in it. The thickness of the substrate is around 100 μm.
In the experiments using the SCAR/WAVE mutants, the substrate used is a polyacrylamide gel of 5% acrylamide and 0.06% bis-acrylamide coated with 0.25 mg/ml collagen [55 (link)] [56 (link)]. The gels consist of two layers: the bottom layer contains no beads, and the upper one contains 4 μl of 2% carboxylate yellow latex beads of 0.1 μm diameter (FluoSpheres; Molecular Probes, Eugene, OR). The thickness of the gel is approximately 40 μm. To functionalize the surface of the gel and transform it to be physiologically compatible to live cells, we coat the surface of the gel with Type II collagen through coupling to the polyacrylamide gel with 1 mM sulfo-SANPAH activated by using UV light.
Figure 1a shows a sketch of the experimental configuration. Figure 1e shows an image of the substrate embedded with beads acquired with a microscope. Similar images to Figure 1e are used to calculate the deformation exerted by the migrating cells over the substrate.
Álvarez-González B., Bastounis E., Meili R., del Álamo J.C., Firtel R, & Lasheras J.C. (2014). Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion. Applied Mechanics Reviews, 66(5), 0508041-05080414.
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4

Nanoparticle Exclusion and Penetration Assays

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Particle exclusion assays were performed by adding red 200 nm FluoSpheres (carboxylate-modified Molecular Probes, Inc., catalog number: F8810) to a final concentration of 0.7% w/v along with fluorescent dextran (Molecular Probes, Inc. Alexa Fluor 647, 10 kDa) to a final concentration of 33 µg/mL. Nanoparticle penetration was investigated using the red, 200 nm FluoSpheres, green 100 nm FluoSpheres (Catalog number: F8803), and green 20 nm FluoSpheres (Catalog number: F8787). When the grafting surface needed to be labeled, 0.007% w/v of the green, 20 nm nanoparticles were added. For details of planar brush height analysis using 200 nm particles, see Supplementary Note 3 and Supplementary Fig. 6.
Wei W., Faubel J.L., Selvakumar H., Kovari D.T., Tsao J., Rivas F., Mohabir A.T., Krecker M., Rahbar E., Hall A.R., Filler M.A., Washburn J.L., Weigel P.H, & Curtis J.E. (2019). Self-regenerating giant hyaluronan polymer brushes. Nature Communications, 10, 5527.
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5

Measuring Myocardial Infarct Size

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Myocardial infarct size was measured as previously described.19 After thoracotomy, the left anterior descending artery was ligated, and 50 μL of green fluorescent FluoSpheres (Molecular Probe, Carlsbad, CA) were injected into the LV of the heart to delineate the area at risk (FluoSpheres‐negative area). After 24 hours, excised hearts were cut into ≈1‐mm‐thick slices from apex to base and then incubated in 2,3,5‐triphenyltetrazolium chloride (Sigma‐Aldrich, St. Louis, MO) solution. Afterward, all slices were placed in a 10% (v/v) formaldehyde solution to improve contrast between stained (viable) and unstained (necrotic) tissues. The infarction area and area at risk were determined of each slice using ImageJ (National Institutes of Health, Bethesda, MD).
Ai W., Bae S., Ke Q., Su S., Li R., Chen Y., Yoo D., Lee E., Jon S, & Kang P.M. (2021). Bilirubin Nanoparticles Protect Against Cardiac Ischemia/Reperfusion Injury in Mice. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 10(20), e021212.
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6

Fluorescent Particle Uptake Assay

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Yellow-green 1μM sulfate treated FluoSpheres (Life Technologies, Grand Island, NY) were coated with recombinant FleA, added to RAW264.7 cells in the presence or absence of 500mM fucose and incubated with agitation for 2 hours at 37°C prior to extensive washing with DMEM to remove unattached FluoSpheres from the cell surface. Cells were fixed in 4% paraformaldehyde prior to analysis on a Becton Dickenson FACSCalibur and FlowJo software (TreeStar, Ashland, OR).
Kerr S.C., Fischer G.J., Sinha M., McCabe O., Palmer J.M., Choera T., Yun Lim F., Wimmerova M., Carrington S.D., Yuan S., Lowell C.A., Oscarson S., Keller N.P, & Fahy J.V. (2016). FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection. PLoS Pathogens, 12(4), e1005555.
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7

Characterization of Carboxylated and Sulfated Latex Nanoparticles

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Carboxylate-modified polystyrene latex 0.02 µm FluoSpheres® (carboxylated latex particles; CLPs) and sulfate polystyrene latex 0.02 µm FluoSpheres® (sulfate latex particles; SLPs; Life Technologies) were suspended in sodium chloride (NaCl) or calcium chloride (CaCl2) at 1 or 10 mM ionic strength (IS) and a pH of 7 ± 0.05. CLP and SLP suspensions were prepared from a stock, which was bath sonicated (Fisher Scientific FS140H, 42±6 Hz) for 2 min before dilution to 20 mg/L (~2.62×10 12 particles/mL). The final suspension was then vortexed at maximum speed for 30 s to homogenize the diluted stock NPs in the suspending buffer. Suspensions were prepared 20 min before characterization or injection into the column.
Mitzel M.R., Sand S., Whalen J.K, & Tufenkji N. (2016). Hydrophobicity of biofilm coatings influences the transport dynamics of polystyrene nanoparticles in biofilm-coated sand. Water research, 92.
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8

Phagocytosis Assay for iPSC-derived Macrophages

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In vivo phagocytosis in iPSC-derived macrophages grown on glass coverslips was detected using 1 μm polystyrene yellow-green FluoSpheres (Themo Fisher) as described in.41 A total of 5 μL of FluoSpheres was directly added to macrophage maturation media (107 beads/mL/105 cells) and incubated for four different time intervals (0.5 h, 1 h, 2 h, and 4 h). After incubation, the cells were washed twice with PBS, fixed with 4% PFA/PBS and imaged using EVOS FL fluorescent microscope (40× objective). 10–15 images/time point/cell line were taken and analysed by two raters in three independent experiments.
Szigeti K., Ihnatovych I., Notari E., Dorn R.P., Maly I., He M., Birkaya B., Prasad S., Byrne R.S., Indurthi D.C., Nimmer E., Heo Y., Retfalvi K., Chaves L., Sule N., Hofmann W.A., Auerbach A., Wilding G., Bae Y, & Reynolds J. (2024). CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization. eBioMedicine, 103, 105093.
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9

Validation of Biomimetic Adhesive Crosslinks

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To validate the presence of physical crosslinks by hydrogen bonds, the barnacle-inspired paste without NHS ester (to avoid effect of covalent crosslinks) was incubated in DMEM with fluorescent microbeads without functional group, coupled with carboxylic acid group, and coupled with primary amine group (FluoSpheres™, Thermo Fisher Scientific) for 30 s in room temperature. Then, the samples were thoroughly washed with clean PBS to remove non-adhered fluorescent microparticles.
To validate the presence of covalent crosslinks by imide bonds, the barnacle-inspired paste without and with NHS ester was incubated in DMEM with fluorescent microbeads coupled with primary amine group (FluoSpheres™, Thermo Fisher Scientific) for 10 min in room temperature. Then, the samples were thoroughly washed with 0.5 M sodium bicarbonate solution to remove fluorescent microparticles adhered by hydrogen bonds43 . The presence of the adhered microbeads was characterized by using a fluorescence microscope (LV10, Nikon) and the number of the adhered microbeads was counted by using Image-J (version 2.1.0).
Yuk H., Wu J., Sarrafian T.L., Mao X., Varela C.E., Roche E.T., Griffiths L.G., Nabzdyk C.S, & Zhao X. (2021). Rapid and coagulation-independent haemostatic sealing by a paste inspired by barnacle glue. Nature biomedical engineering, 5(10), 1131-1142.
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10

Characterization of Laser Beam Profiles

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To measure the beam profiles at the focal plane of DO lens, the sample chamber was filled with 7 μg/ml sulforhodamine B in deionized water, and fluorescent signals were imaged by the CMOS camera. To estimate the beam extent and thickness, FWHMs along the longitudinal ( x ) and transverse ( y ) axis were calculated. For resolution evaluation experiments, yellow–green fluorescence beads (FluoSpheres, ThermoFisher) with a 200-nm diameter embedded in 1% agarose gel were used. PSFs were calculated by analyzing the z-stack images of the beads using the software PSFj (http://www.knoplab.de/psfj/), which resulted in the FWHM values of the xy -plane and z -axis. For the laser power determination experiment, yellow–green fluorescence beads (FluoSpheres, ThermoFisher) with a 2-μm diameter embedded in 1% agarose gel were used. Using a maximum-intensity projection view of the z-stack images, centroid intensities of the beads were measured using the software PSFj. In all these experiments, a laser (InSight Deepsee) with a 925-nm wavelength was used.
Takanezawa S., Saitou T, & Imamura T. (2021). Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination. Nature Communications, 12, 2979.
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