For each test, we used the recommended sample volume of 100 μL in a 1.5 mL microcentrifuge tube. PBS is at an optimal salt concentration for appropriate conductivity that is required for accurate counting performance. The concentration range tested (50,000 to 1,500,000 beads/mL), corresponds to the upper and lower limits of detection for the 40 μm sensor. Materials and Methods Bead counting using the Scepter™ cell counterīead suspensions ( Table 2 for list of beads tested) were serially diluted in phosphate-buffered saline (PBS, Product No. While the Scepter™ device was initially optimized for cell counting, herein we report that the device is also well suited for the precise counting of diverse types of beads commonly employed in other biological applications. The 40 μM and 60 μM aperture sensors are engineered with a microfabricated sensing zone that enables discrimination by bead size and bead volume at submicron and subpicoliter resolution, respectively Table 1 outlines the specifications for each sensor type.
The instrumentation has been collapsed into a device the size of a pipette, and uses a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. With the Scepter™ cell counter, we have combined the ease of automated instrumentation with the accuracy of impedance-based counting using the Coulter principle in an affordable, handheld tool. 2 For most researchers, a critical barrier to using an automated vision-based or flow-based system is the price associated with large benchtop instruments. By precisely controlling the flow, volumetric measurements can be obtained, thereby permitting estimation of sample bead concentration. 1 Flowbased devices measure particles in a stream using impedance-based detection.
Most vision-based counters use the standard Trypan blue exclusion assay to assess viability, and employ a digital camera with image analysis software to determine particle size and concentration.
Automated counting devices are commercially-available in two formats: vision-based platforms and flow-based systems.
While manual counting using a hemocytometer is accessible and inexpensive, it is laborious and error-prone due to user subjectivity. Accurate determination of bead counts at the onset of each assay allows for standardization of bead concentration across multiple samples, and minimizes errors and variation in downstream results.Ī number of methodologies are currently available for particle counting. In bead-based multiplexed immunoassays, these particles are coated with unique recognition molecules, such as epitope-specific antibodies, permitting capture and precise quantification of desired analyte(s). Depending on the nature of the assay, the beads may either possess magnetic properties or be labeled internally with a fluorescent dye. Micron-sized beads are used in a variety of biological applications, ranging from daily validation of flow cytometer performance, to purification of fusion protein constructs from cell lysates.