Three sections will be discussed: Instrument set-up for small particles, bead resolution, and microparticle sorting (erythrocyte, endothelial and platelet).
Methods: Instrument Set-up: To be able to detect particles smaller than 1 μm, particles residing in the sheath and sample lines were eliminated by using glass 5 mL sample tubes, 0.1 μm inline sheath filter, 0.2 μm air filter, 0.2 μm pre-filtered sample buffer and cleaning both the sheath and sample lines with 10% Sodium hypochlorite (bleach). Bead resolution: Beads were mixed in equivalent concentrations and run using the new FSC optics at speeds of 100 eps. Microparticles: Microparticles for erythrocytes and platelets were harvested from 18 mL human blood, centrifuged 1x at 5K rpm for 15 mins and the supernatant centrifuged in microfuge tubes at the maximum speed (16K rpm) for 20 mins. The pellet was stained with Annexin-V FITC as per standard procedure and stained with additional antibodies for 20 min at RT in the dark. Antibodies: Platelets Mouse Anti-Human CD41-PC5, CD61-PC7, CD42b-PE, Annexin-V FITC, CD45 PB; Erythrocytes: Mouse Anti-Human CD235a-PC7, Annexin-V FITC, CD45-PB Endothelial: CD105-PE, CD146-PC5, Annexin-V FITC, CD45-PB. Flow Cytometry: Bead particles and microparticles were triggered on FSC, SSC and fluorescent parameters. Microparticle PE-tandems were run both on the 488 nm and a modified 561 nm filter set and sorted at 50K eps.
Results: Instrument Set-up: Utilizing the instrument set-up, the noise particles within the system were reduced to less than 0.2 μm on the FSC-H vs SSC-H histogram plots. Bead Resolution: The 0.2 and 0.3 μm beads were resolvable with a 0.1% overlap between populations and were visible above background noise. Additionally, the enhanced FSC measured beads, sized 0.2 to 30 μm to be resolved concurrently. Microparticles: Erythrocyte and platelet microparticles were visible from FSC noise and sorted on positive markers.