Microspheres - Section 6.5

Fluorescent FluoSpheres and TransFluoSpheres Microspheres

Molecular Probes' intensely fluorescent FluoSpheres (Working with FluoSpheres(R) Fluorescent Microspheres) and TransFluoSpheres (TransFluoSpheres(R) Fluorescent Microspheres) beads are manufactured using high-quality, ultraclean polystyrene microspheres. These microspheres are internally labeled with Molecular Probes' proprietary dyes, making them the brightest fluorescent microspheres available (Molecular Probes' yellow-green-fluorescent FluoSpheres beads compared with other commercially available yellow-green-fluorescent microspheres - Table 6.5). We employ methods to ensure that each bead is heavily loaded with dye. The protective environment within the bead matrix shields the dyes from many of the environmental effects that cause photobleaching of exposed fluorophores.

The stability, uniformity and reproducibility of fluorescent microspheres, as well as the extensive selection of colors available make our microspheres the preferred standards for research and diagnostic assays that use fluorescence. We have developed several important microsphere-based products for calibrating and aligning fluorescence microscopes (Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1) and flow cytometers (Flow Cytometry Reference Standards - Section 23.2). By carefully selecting dyes that can be incorporated within the microspheres, our CompenFlow microspheres duplicate the spectra of widely used fluorophores such as fluorescein (Figure) or R-phycoerythrin (Figure) that are not themselves soluble in polystyrene beads. Not only are our yellow-green–fluorescent beads more photostable, but their emission spectra are not affected by changes in pH, as are conventional fluorescein-labeled microspheres. Fluorescent microspheres can be fixed in formalin and embedded in paraffin if care is used to avoid extraction of the noncovalently associated dyes from the microspheres.

We also offer fluorescent microspheres conjugated to biotin, streptavidin and NeutrAvidin biotin-binding protein, which are described below. Discounts for large-volume orders are available, as well as custom preparation of microspheres with other colors, sizes and surface coatings; please contact our Custom and Bulk Sales Department for more information. The FluoSpheres and TransFluoSpheres beads, their conjugates and most of our microsphere-based standards for fluorescence microscopy and flow cytometry are covered by Patents. These products are offered for research purposes only. Molecular Probes welcomes inquiries about Licensing these products for resale or other commercial uses.

Colored and Unstained Microspheres

In addition to our extensive line of fluorescent microspheres, Molecular Probes now offers a wide selection of colored and unstained microspheres for research applications as well as for water- and air-flow testing and bead-based diagnostic applications. Through its acquisition of Interfacial Dynamics Corporation (IDC), Molecular Probes can provide milliliter to 500-liter quantities of ultraclean microspheres with diameters from 20 nm to 10.0 µm and with more than 20 different surface functionalities; see http://www.idclatex.com for a complete listing of the colored and unstained microspheres available. IDC pioneered the commercial development of surfactant-free polymer particles used in bead-based assay systems (see below for a description of Colloids and Interfaces with Surfactants and Polymers — An Introduction, by Jim Goodwin, C31200) and has been a key supplier of beads for Molecular Probes' fluorescent microspheres. IDC's outstanding capabilities in microsphere manufacturing allow a high level of control of the colloid engineering employed in the particle synthesis, providing unbeatable batch-to-batch consistency. Microsphere manufacturers often use surfactants to prevent aggregation. However, standards of surfactant purity are generally not very high, leading to an < particle surface and variable protein attachment. With the ultraclean microspheres manufactured by IDC, and now Molecular Probes, no surfactants are required to prevent aggregation, taking much of the guesswork out of stability and adsorption experiments. We can tailor-make colored and unstained microspheres of many sizes, surface chemistries, densities and volumes to meet the diverse needs of customers, including academic, industrial and government laboratories, as well as major global diagnostic companies; please contact our Custom and Bulk Sales Department for more information.

Fluorescent microspheres have been used as immunofluorescent reagents, retrograde neuronal tracers, microinjectable cell tracers (FluoSpheres and TransFluoSpheres Microspheres for Tracing - Section 14.6) and standardization reagents for microscopy (Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1) and flow cytometry (Flow Cytometry Reference Standards - Section 23.2). Arrays of fluorescent microspheres that differ in intensity, size or excited-state lifetime are extensively used for simultaneous assays to determine multiple analytes in a single sample. Furthermore, fluorescent microspheres have been employed to:

• Determine blood flow in tissues (FluoSpheres and TransFluoSpheres Microspheres for Tracing - Section 14.6)
• Develop high-resolution maps of regional pulmonary ventilation (FluoSpheres and TransFluoSpheres Microspheres for Tracing - Section 14.6)
• Measure retinal and choroidal circulation
• Define the functional diameter of alveolar microvessels using microspheres of increasing diameters
• Measure the blood velocity in tumor vasculature in vivo
• Investigate phagocytic processes (Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1)
• Detect low-abundance receptors
• Probe specific sequences for protein binding on single DNA molecules
• Develop a simultaneous assay for digoxin and theophylline
• Follow the fate of transplanted cells, such as donor erythrocytes, in patients who received allogenic bone marrow transplants
• Quantitate lymphocyte numbers in a lymphocyte–endothelial adhesion/transendothelial migration assay
• Image three different Candida albicans antigens simultaneously
• Investigate binding mechanisms of neural cell adhesion molecules
• Track the lateral mobility of GPI-anchored proteins in supported bilayers, Thy1 molecules in the plasmalemma of live fibroblasts and surface receptors during cell division
• Detect amines on the surface of self-assembled monolayers of a microfabricated device
• Make kinesin force measurements with optical tweezers
• Analyze the elasticity of single DNA molecules via optical trapping
• Simultaneously screen for multiple analytes by flow cytometry
• Quantitate adenovirus using a flow microsphere immunoassay (FMIA)
• Detect binding of a fluorescent ligand to its receptor using microvolume fluorometry

In addition, fluorescent microspheres are potentially more sensitive than colorimetric methods in most, if not all, of the major microsphere-based diagnostic test systems presently in use, including latex-agglutination tests, filter-separation tests, particle-capture ELISA methods and two-particle sandwich techniques.

A Wide Array of Fluorescent Colors

Molecular Probes' FluoSpheres fluorescent microspheres contain dyes with excitation and emission wavelengths that cover the entire spectrum from the near UV to the near-infrared. Figure 6.45 shows the normalized emission spectra for 10 fluorescent colors of FluoSpheres beads. Because long-wavelength (>680 nm) light can penetrate tissues, our far-red– and infrared-fluorescent microspheres may allow researchers to conduct experiments that were not previously possible with beads that emit at shorter wavelengths. We would like to highlight the following FluoSpheres products:

• Our blue-fluorescent FluoSpheres beads with excitation/emission maxima of 350/440 nm contain an improved blue-fluorescent dye that provides superior brightness and a longer shelf life. We also offer blue-fluorescent FluoSpheres beads with slightly shorter-wavelength fluorescence spectra (excitation/emission maxima ~365/415 nm).
• Our yellow-green–fluorescent FluoSpheres beads have excitation/emission maxima of 505/515 nm and thus are excited very efficiently using the 488 nm spectral line of the argon-ion laser, resulting in exceptionally intense fluorescence (Molecular Probes' yellow-green-fluorescent FluoSpheres beads compared with other commercially available yellow-green-fluorescent microspheres - Table 6.5).
• Our orange-, red-orange– and red-fluorescent FluoSpheres beads have excitation maxima of 540 nm, 565 nm and 580 nm, respectively.
• The nile red–fluorescent FluoSpheres beads have broad excitation/emission bandwidths at 535/575 nm, making them compatible with filter sets appropriate for fluorescein, rhodamine and Texas Red dyes.
• Our crimson- and dark red–fluorescent FluoSpheres beads with excitation/emission maxima of 625/645 nm and 660/680 nm, respectively, are efficiently excited by the 633 nm spectral line of the He–Ne laser. Although the dark red–fluorescent beads are significantly less fluorescent than the crimson-fluorescent particles, they fluoresce at wavelengths that are longer than, and clearly distinguishable from, those of the crimson-fluorescent particles.
• The far-red–fluorescent FluoSpheres beads with excitation/emission maxima of 690/720 nm are compatible with diode lasers — inexpensive excitation sources that are increasingly being used in fluorescence instrumentation. These far-red–fluorescent beads may also prove useful for making direct fluorescence measurements in autofluorescent materials such as blood, plant tissues and marine organisms.
• The infrared-fluorescent FluoSpheres beads with excitation/emission maxima of 715/755 nm are the longest-wavelength fluorescent microspheres currently available from any source. These beads absorb and emit at wavelengths at which most tissues are almost optically transparent.
• Our europium luminescent and platinum luminescent FluoSpheres beads have excitation/emission maxima of 340–370/610 nm (Figure 6.47) and ~390/650 nm (Figure 6.48), respectively, and decay times of >40 microseconds for the platinum microspheres and >600 microseconds for the europium microspheres, far longer than those of conventional fluorescent probes and autofluorescent samples. The beads should be useful as standards for time-resolved microscopy and for tracing applications in highly autofluorescent samples.

Our FluoSpheres beads are many times brighter than fluorescent microspheres from other companies (Molecular Probes' yellow-green-fluorescent FluoSpheres beads compared with other commercially available yellow-green-fluorescent microspheres - Table 6.5). Fluorescein equivalents in our yellow-green-fluorescent FluoSpheres beads - Table 6.6 shows the approximate number of unquenched fluorescein equivalents in our yellow-green–fluorescent FluoSpheres beads. The intensity of the beads is sufficient to allow visualization of single particles, even for our smallest microspheres, which appear as point sources (); see the description of our PS-Speck Microscope Point Source Kit (P7220) in Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1. Moreover, aqueous suspensions of FluoSpheres beads do not fade appreciably when illuminated by a 250-watt xenon-arc lamp for 30 minutes. Indeed, most of our FluoSpheres beads show little or no photobleaching, even when excited with the intense illumination required for fluorescence microscopy.

Although some of our FluoSpheres beads are available in limited sizes, colors and surface functions, we will prepare custom orders upon request. Molecular Probes has considerable experience developing standards, including microsphere-based standards for companies selling fluorescence instrumentation. Additional sizes and colors of these labeled microspheres can be custom-ordered through our Custom and Bulk Sales Department. FluoSpheres beads can also be prepared with intensities that are lower than those of our regular products — a desirable feature in some multicolor applications. FluoSpheres beads with calibrated intensities are already offered in our InSpeck Microscope Intensity Calibration Kits (Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1) and LinearFlow Flow Cytometry Intensity Calibration Kits (Flow Cytometry Reference Standards - Section 23.2), which are each available in several fluorescent colors. Molecular Probes offers a variety of other microsphere reference standards designed to facilitate adjustment and calibration of fluorescence microscopes (Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1) and flow cytometers (Flow Cytometry Reference Standards - Section 23.2).

A Wide Range of Sizes

To meet the diverse needs of our customers, we offer FluoSpheres beads in a variety of sizes (Summary of Molecular Probes' FluoSpheres fluorescent microspheres - Table 6.7). The smallest microspheres are currently about 0.02 µm in diameter, with a coefficient of variation (CV) of about 20% as determined by electron microscopy. The size uniformity improves with increasing size, with the CV decreasing from ~5% for 0.1 µm FluoSpheres beads to ~1% for those with 10–15 µm diameters. The sizes specified in the product names are nominal bead diameters; because of batch-to-batch variation in the undyed microspheres, the actual mean diameters shown on the product labels may differ from the nominal diameters, especially for the smaller microspheres. Because of their small size, 0.02–0.04 µm microspheres are effectively transparent to light in aqueous suspensions and behave very much like true solutions.

Four Different Surface Functional Groups

We prepare FluoSpheres beads with four different surface functional groups, making them compatible with a variety of conjugation strategies. Our fluorescent dyes have negligible effect on the surface properties of the polystyrene beads or on their protein adsorption. We caution, however, that the surface properties have an important role in the functional utility of the microspheres; we cannot guarantee the suitability of a particular bead type for all applications.

• Carboxylate-modified FluoSpheres beads have pendent carboxylic acids, making them suitable for covalent coupling of proteins and other amine-containing biomolecules using water-soluble carbodiimide reagents such as EDAC (E2247, Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3). In order to both decrease nonspecific binding and provide additional functional groups for conjugation, these FluoSpheres beads have a high density of carboxylic acids on their surfaces.
• Sulfate FluoSpheres beads are relatively hydrophobic particles that will passively and nearly irreversibly adsorb almost any protein, including albumin, IgG, avidin and streptavidin.
• Aldehyde–sulfate FluoSpheres beads, which are sulfate microspheres that have been modified to add surface aldehyde groups, are designed to react with proteins and other amines under very mild conditions.
• Amine-modified FluoSpheres beads can be coupled to a wide variety of amine-reactive molecules, including the succinimidyl esters and isothiocyanates of haptens and drugs or the carboxylic acids of proteins, using a water-soluble carbodiimide. The amine surface groups can also be reacted with SPDP (S1531, Chemical Crosslinking Reagents - Section 5.2) to yield (after reduction) microspheres with pendent sulfhydryl groups.

Fluorescent Microspheres Conjugated to Biotin, Avidin and Streptavidin

Molecular Probes offers yellow-green–fluorescent microspheres conjugated to biotin or streptavidin, and yellow-green–fluorescent, red-fluorescent, europium luminescent, platinum luminescent and nonfluorescent microspheres conjugated to NeutrAvidin biotin-binding protein (Summary of biotin-, streptavidin- and NeutrAvidin biotin-binding protein-labeled FluoSpheres microspheres - Table 6.8). NeutrAvidin biotin-binding protein has been specially processed to remove carbohydrates and lower the isoelectric point, resulting in a near-neutral protein that has significantly lower nonspecific binding than conventional avidin. These microsphere conjugates provide our customers with valuable tools for improving the sensitivity of flow cytometry applications and immunodiagnostic assays. They may also be useful as tracers that can be detected with standard enzyme-mediated avidin/streptavidin methods. Additional sizes and colors of these microspheres can be custom-ordered through our Custom and Bulk Sales Department.

Fluorescent Microspheres Coated with Collagen

Fibroblasts phagocytose and then intracellularly digest collagen. These activities play an important role in the remodeling of the extracellular matrix during normal physiological turnover of connective tissues, in development, in wound repair and possibly in aging and various disorders. A well-established procedure for observing collagen phagocytosis by either flow cytometry or fluorescence microscopy involves the use of collagen-coated fluorescent microspheres, which attach to the cell surface and become engulfed by fibroblasts. Molecular Probes offers yellow-green–fluorescent FluoSpheres collagen I–labeled microspheres in either 1.0 µm or 2.0 µm diameter (F20892, F20893; FluoSpheres(R) Collagen I–Labeled Microspheres) for use in these applications. These microspheres have collagen I from calf skin attached covalently to their surface.

Europium and Platinum Luminescent Microspheres for Time-Resolved Fluorometry

Detecting low levels of protein or DNA targets in a tissue sample or on a membrane using classic fluorochromes is sometimes difficult and prone to errors because specific fluorescence signals tend to be low and are usually mixed with nonspecific signals and autofluorescence. One approach to improve detectability is the use of time-resolved luminescence reagents, such as our FluoSpheres europium luminescent microspheres (FluoSpheres(R) Europium Luminescent Microspheres) and FluoSpheres platinum luminescent microspheres (FluoSpheres(R) Platinum Luminescent Microspheres). The FluoSpheres europium luminescent beads contain Eu³⁺ coordination complexes with luminescence decay times of >600 microseconds — much longer that the <50 nanosecond decay time of conventional fluorophores and autofluorescence. The luminescence of the Pt²⁺ chelate in the FluoSpheres platinum luminescent microspheres has a decay time of >40 microseconds. Thus, time-gated fluorescence detection using these microspheres results in complete rejection of autofluorescence signals. In addition, both the europium luminescent and platinum luminescent microspheres feature long-wavelength emissions (610–650 nm) that are well separated from their excitation peaks (340–390 nm) (Figure 6.47, Figure 6.48). Because of this exceptionally large Stokes shift, filter combinations can be chosen that effectively isolate the desired luminescence signal.

These microspheres are available uncoated (F20880, F20881, F20882, F20886, F20887, F20888) or conjugated to NeutrAvidin biotin-binding protein (F20883, F20884, F20885, F20889, F20890, F20891), with nominal diameters of 0.04 µm, 0.2 µm or 1.0 µm. Beads labeled with NeutrAvidin biotin-binding protein can be used for the indirect detection of antigens and DNA targets in many biotin/avidin-based assays.

For applications requiring several different microsphere colors or sizes, we offer three types of fluorescent microsphere starter kits:

• The FluoSpheres Fluorescent Color Kit (F10720) consists of 1 mL samples of yellow-green–, orange-, red- and dark red–fluorescent, carboxylate-modified 0.04 µm FluoSpheres beads packaged as high-density, azide-free suspensions for microinjection.
• FluoSpheres Size Kits contain 1 mL samples of carboxylate-modified FluoSpheres beads in 0.02, 0.1, 0.2, 0.5, 1.0 and 2.0 µm sizes. These beads are available in yellow-green– (F8888) or red- (F8887) fluorescent colors.
• FluoSpheres Blood Flow Determination Fluorescent Color Kits provide several different fluorescent colors of our 10 µm (F8890) or 15 µm (F8891, F8892, F21015) FluoSpheres polystyrene microspheres (FluoSpheres and TransFluoSpheres Microspheres for Tracing - Section 14.6).

Molecular Probes' Constellation microspheres for imaging (C14837) can be used to demonstrate hands-on techniques with a fluorescence microscope. Constellation microspheres consist of a selected mixture of beads in assorted sizes and colors (, ) that can be used to practice adjusting the focus and switching filters on a fluorescence microscope. These microspheres are stable at room temperature, so they can be conveniently stored.

Advantages of TransFluoSpheres Fluorescent Microspheres

Molecular Probes' Patented TransFluoSpheres fluorescent microspheres (Summary of Molecular Probes' TransFluoSpheres fluorescent microspheres - Table 6.9; Figure 6.49, ) are specially designed to overcome the limitations imposed by modern fluorescence instrumentation. Many flow cytometers, confocal laser-scanning microscopes and laser scanners incorporate the argon-ion laser as the excitation source, thereby limiting the available excitation wavelengths to the laser's 488 nm and 514 nm spectral lines and severely restricting simultaneous multicolor detection. Ideally, it would be useful to have a series of fluorescent dyes with absorption maxima close to the argon-ion laser's spectral lines, but with emission maxima at a variety of longer wavelengths. This approach would require that some of the dyes exhibit large Stokes shifts — defined as the separation of the absorption and emission maxima. Unfortunately, very few low molecular weight dyes have a combination of a large Stokes shift and a high molar absorptivity (Limitations of Low Molecular Weight Dyes - Note 6.2 ). For example, the Texas Red fluorophore — often used in combination with fluorescein — has a particularly low absorption at 488 nm and 514 nm. In applications that employ the argon-ion laser as an excitation source, Texas Red conjugates have a low fluorescence output that is easily obscured by the more intense fluorescein fluorescence, even when detected past 600 nm.

Our TransFluoSpheres beads, which incorporate two or more fluorescent dyes that undergo excited-state energy transfer, exhibit Stokes shifts that can be extremely large. Each microsphere contains a dye with an excitation peak that maximally overlaps the spectral output of a commonly used excitation source (for example, the 488 nm spectral line of the argon-ion laser; Figure 6.50). In addition, each microsphere contains one or more longer-wavelength dyes that are carefully chosen to create a relay series that can efficiently transfer the energy from the initially excited dye to the longest-wavelength acceptor dye. The proprietary dyes used in the TransFluoSpheres beads are optimally loaded to ensure that the excitation energy is efficiently transferred from dye to dye so that essentially only the longest-wavelength dye in the series exhibits significant fluorescence. Because these TransFluoSpheres beads fluoresce at a considerably longer wavelength than the excitation wavelength, they provide a signal that can be detected in samples with significant Rayleigh or Raman scattering or with endogenous fluorescent compounds such as bilins, flavins and certain drugs. Also, the large Stokes shifts exhibited by the TransFluoSpheres beads allow the use of broadband filters, both to excite the sample and to detect the emission, resulting in a greater fluorescent signal (Figure 6.49).

Figure 6.49 Schematic diagram of the advantages of the large Stokes shift exhibited by our TransFluoSpheres beads. A1 and E1 represent the absorption and emission bands of a typical TransFluoSpheres bead. The large separation of the absorption and emission maxima (Stokes shift) is characteristic of our TransFluoSpheres beads. Unlike most low molecular weight fluorescent dyes, which show considerable overlap of their absorption and emission spectra, the TransFluoSpheres beads can be excited (EX) across the entire absorption band A1 and the resulting fluorescence can be detected across the full emission band E1, thereby allowing the researcher to maximize the signal (S1). Moreover, because of the large Stokes shifts of the TransFluoSpheres beads, researchers can often avoid problems associated with autofluorescence. The absorption and emission bands of a typical autofluorescent component are represented in this Figure .y A2 and E2. Although the endogenous fluorescent species will be excited simultaneously with the TransFluoSpheres beads, the resulting emission (E2) does not coincide with E1 and is therefore readily rejected by suitably chosen optical filters.

Figure 6.50 Fluorescence emission spectra of our five 488 nm light–excitable TransFluoSpheres beads, named according to their excitation/emission maxima (nm): A) 488/560, B) 488/605, C) 488/645, D) 488/685 and E) 488/720. The arrow in each spectrum represents the 488 nm spectral line of the argon-ion laser.

TransFluoSpheres Beads to Match Different Excitation Sources

Molecular Probes offers TransFluoSpheres beads that are compatible with several different excitation sources:

• Argon-ion laser–excitable TransFluoSpheres beads (Figure 6.50) that have an excitation maximum near 488 nm but emit at 560 nm (T8864, T8872, T8880), 605 nm (T8865), 645 nm (T8867, T8883), 685 nm (T8868, T8876) or 720 nm (T8869)
• Red He–Ne laser–excitable TransFluoSpheres beads with excitation/emission maxima of 633/720 nm (T8870, T8878)
• Green He–Ne laser–excitable TransFluoSpheres beads with excitation/emission maxima of 543/620 nm (T8874)

The series of TransFluoSpheres beads with 488 nm excitation enables researchers to potentially detect five experimental parameters simultaneously. TransFluoSpheres beads can also be combined with our more traditional FluoSpheres beads or with low molecular weight dyes for multicolor detection. Using carbodiimide reagents such as EDAC (E2247, Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3), researchers can couple protein or other amine-containing molecules to our carboxylate-modified TransFluoSpheres beads, making these microspheres suitable for a wide range of applications. TransFluoSpheres beads can be used in the major microsphere-based diagnostic test systems and in experiments that currently employ standard fluorescent microspheres to measure regional blood flow (T13090, FluoSpheres and TransFluoSpheres Microspheres for Tracing - Section 14.6), study phagocytosis (Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1), detect cell-surface antigens and trace neurons.

We offer TransFluoSpheres microspheres with excitation/emission maxima of 488/605 nm (T8860, T8861) conjugated to NeutrAvidin biotin- binding protein, as well as TransFluoSpheres beads with excitation/emission maxima of 488/645 nm (T10711) conjugated to streptavidin. Flow cytometry studies demonstrate that the sensitivity of our 40 nm TransFluoSpheres beads conjugated to streptavidin is superior to that of fluorescein streptavidin and comparable to that of R-phycoerythrin–streptavidin for detecting biotinylated epidermal growth factor (EGF) bound to EGF receptors. In multicolor experiments, the long-wavelength fluorescence emission of these TransFluoSpheres beads permits their use simultaneously with fluorescein- and R-phycoerythrin–labeled probes. For all applications requiring protein-coated microspheres, we strongly recommend using our BlockAid blocking solution (B10710) to reduce nonspecific binding.

If wavelength or bead-size requirements are not met by our current selection of products, we invite inquiries about custom synthesis by contacting our Custom and Bulk Sales Department. Molecular Probes can also fine-tune the excitation and emission of our microspheres to match a researcher's needs. In addition, we can covalently conjugate our TransFluoSpheres beads to other target-specific proteins to provide detection reagents that have potentially greater sensitivity in flow cytometry applications and immunodiagnostic assays.

Molecular Probes' intensely fluorescent and highly photostable FluoSpheres and TransFluoSpheres microspheres have significant potential for applications requiring probes that can deliver a strong signal. Unfortunately, microspheres conjugated to proteins have hydrophobic regions that may cause them to bind to nontarget surfaces in some experimental systems. This nonspecific binding can often be relieved by the use of a blocking solution. However, we have found that microspheres require a stronger blocking solution than those in common use, and therefore we have developed the BlockAid blocking solution (B10710, BlockAid Blocking Solution).

Our BlockAid reagent is a protein-based blocking solution designed for use with TransFluoSpheres and FluoSpheres microspheres conjugated to biotin, streptavidin, NeutrAvidin biotin-binding protein or other proteins. In our tests, BlockAid blocking solution was mixed with FluoSpheres microspheres conjugated to streptavidin, which were then used to stain several different cell types for subsequent analysis by flow cytometry. We found the BlockAid blocking solution to be superior to blocking solutions available from other companies, as well as to several standard blocking solutions described in the scientific literature for reducing nonspecific binding of labeled microspheres. BlockAid blocking solution has been found to be effective in flow cytometry applications involving NIH 3T3, A431, RAW and Jurkat cell lines; however, with the HMC-1 cell line, it did not appear to offer any advantages over standard blocking solutions. We expect that BlockAid blocking solution will be useful for reducing the nonspecific binding of protein-coated or other macromolecule-coated microspheres in a variety of flow cytometry and microscopy applications.

Our microsphere bibliography (Bibliography for M8997) contains over 1200 references, and continuously updated copies are available upon request from our Technical Assistance Department or through this web site. This bibliography includes references in which microspheres from several different sources were used in a wide variety of applications. Because the source, surface properties and size of the microspheres may affect the application, the methods described in these references should be considered guides rather than definitive protocols. In particular, the bibliography lists several references that cite the use of fluorescent microspheres for retrograde tracing, and our FluoSpheres beads have not performed consistently in this application. However, our fluorescent microspheres are guaranteed to be significantly brighter than microspheres available from other sources (Molecular Probes' yellow-green-fluorescent FluoSpheres beads compared with other commercially available yellow-green-fluorescent microspheres - Table 6.5).

We also offer the book Colloids and Interfaces with Surfactants and Polymers — An Introduction (C31200), by J. Goodwin and published in 2004. This introductory text discusses the basic principles of colloid and interface science, with emphasis on recent information about the interactions between particles and the concentrated state. With its nonmathematical approach, this book includes in-depth descriptions of widely used techniques such as rheology, as well as many examples of colloid and interface science, and can be used as a bridge to more specialized texts in this field.