Microspheres Support—Troubleshooting

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. 

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.

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

A variety of conditions can contribute to microsphere aggregation; avoid subjecting your microspheres to the following conditions:

• Freezing temperatures
• Microbial contaminants
• Buffers with high salt concentration
• Extremes of pH
  • Acidic pH will protonate carboxyl and sulfonate groups; basic pH will deprotonate amine groups.
• Heating, excessive vortexing, and excessive sonication 
  • This is particularly relevant for protein-labeled microspheres

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.

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

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.

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 >1 µm in diameter can be centrifuged at 1,300 rpm.