FluoSpheres™ Polystyrene Microspheres, 15 μm, red-orange fluorescent (565/580), for blood flow determination - FAQs

We offer FluoSpheres fluorescent microspheres that are available in 10 and 15 µm diameters, with various wavelengths, for tracer studies. If those sizes or available colors are not optimal for your system, then we recommend the carboxylate-modified microspheres, which are available in a wide range of sizes and colors.

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The warranty period for FluoSpheres microspheres is 1-year from the date of shipment.

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Centrifugation is not an effective way to collect smaller microspheres; many particles remain in the solution even if you can visualize a small pellet. For beads less than 1 µm in diameter, we recommend washing by either:

Cross-flow filtration, as these particles have a very high compression modulus and can withstand high g-forces without risk of harm or dialysis with a 500 kDa MWCO
Note: Microspheres greater than 1 µm in diameter can be centrifuged at 1,300 rpm.

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Bacterial contamination is the most common cause of microspheres becoming unusable. Many of our particles are supplied with a low level of sodium azide to prevent bacterial contamination, but sometimes this can still occur. Bacterial contamination is best assessed by plating on appropriate growth medium and checking the plates after 72 hr.

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Even brief freezing can cause irreversible aggregation and potential distortion of the bead shape. You should not use these microspheres.

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Non-specific binding can often be relieved by a blocking solution, but microspheres seem to require a stronger blocking solution than those most commonly commercially available. Hence, we've developed the BlockAid Blocking Solution (Cat. No. B10710). This reagent is a protein-based blocking solution designed for use with FluoSpheres microspheres and TransFluoSpheres microspheres conjugated to biotin, streptavidin, NeutrAvidin biotin-binding protein, or other proteins. The BlockAid Blocking Solution has proven useful for reducing the nonspecific binding of protein-coated or other macromolecule-coated microspheres in a wide variety of flow cytometry, microscopy, and microarray applications.

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We recommend using a bath sonicator to disperse microsphere aggregates. Do not use a probe sonicator as this will damage the microspheres.

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The foaming is from Tween 20, which is present in the stock solution to help prevent aggregation. It is normal to see bubbles from this reagent.

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Aggregation is usually reversible unless the beads were frozen. Sonicate the microspheres in a bath sonicator to disperse just prior to use. You can also add a small concentration of Tween 20 or Triton X-100 unless you are using the microspheres in a live-cell system. For protein-labeled microspheres like NeutrAvidin microspheres, you should use the gentlest method possible to disrupt aggregates to avoid disrupting the protein conformation. Please be aware that the smaller the microsphere diameter, the more likely it is that aggregation may occur.

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Microspheres greater than 1 µm in diameter can be centrifuged at 1,300 rpm, but once pelleted they may not disperse readily or not at all. Centrifugation is not an effective way to collect smaller microspheres; for beads less than 1 µm in diameter, we recommend washing by either cross-flow filtration or dialysis with a 500 kDa MWCO.

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We offer a variety of FluoSphere microspheres and kits specifically designed for blood flow determination; if the available colors and/or sizes do not suit your needs, we recommend using beads modified with carboxylate groups for blood flow determination applications. We give some basic usage guidelines in the FluoSpheres Fluorescent Micospheres for Tracer Studies product manual (https://tools.thermofisher.com/content/sfs/manuals/mp13080.pdf). Additionally, the University of Washington Seattle offers the Fluorescent Microsphere Resource Center (FMRC) website (http://fmrc.pulmcc.washington.edu/fmrc.shtml) with detailed applications manuals and references for using fluorescent microspheres to evaluate regional blood flow.

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Point spread functions will require a subresolution bead, and we have two different subresolution bead products that could be used for this purpose. TetraSpeck Microspheres, 0.1 µm (Cat. No. T7279), are single subresolution microspheres that are simultaneously stained with blue, green, orange, and dark red dyes. The PS-Speck Microscope Point Source Kit (Cat. No. P7220) consists of individually-stained blue, green, orange, and dark red fluorescent microspheres that are approximately 175 nm in diameter.

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We recommend using a ProLong product or any other aqueous mounting media. You should avoid organic-based mountants such as toluene-based mounting media.

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The FluoSpheres microspheres may have a trace amount of surfactant. Only IDC Latex beads with diameters greater than 100 nm are actually surfactant-free.

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The stability in alcohol or water/alcohol mixtures is dependent on the alcohol chain length. The beads are fairly stable in 40% methanol, but they will dissolve much more readily in long chain alcohols. Prolonged storage of beads in an alcohol containing solution will tend to leach out the dye.

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We recommend using dialysis with a MWCO of approximately 500,000 to remove the sodium azide from the microsphere-containing buffer.

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We do not determine the exact number of dye molecules per microsphere. The number of dye molecules is a function of the diameter of the particle and the properties of the dye.

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The polystyrene microspheres are swelled in a solvent containing a hydrophobic dye specific to the FluoSpheres product. The dye is then able to diffuse into the polystyrene matrix. The beads are then removed from the solvent and dialyzed into an aqueous environment; this reverses the swelling and traps the dye in the polystyrene. Once trapped, the dye is fairly protected from the external environment. The exact protocol and identity of the hydrophobic dyes are proprietary.

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