Characterizing the Light-Scatter Sensitivity of the CytoFLEX Flow Cytometer

George Brittain, Sergei Gulnik, and Yong Chen, Beckman Coulter Life Sciences, Miami, FL 33196

Introduction

Extracellular vesicles (EVs) and other biological nanoparticles (NPs) generally fall within the optical noise of light-scatterbased detection methods, and most flow cytometers are not sensitive enough to effectively detect NPs less than 300nm in diameter. The CytoFLEX is a notable exception to this: it is so sensitive that the SSC detector actually has an attenuation filter to reduce 95% of the scatter signal, adjusting it to a range useful for cells. As an alternative, Violet SSC (VSSC) can be used to bring the CytoFLEX sensitivity well into the nanoparticle range.

In order to better characterize the biological threshold sensitivity of the CytoFLEX using VSSC, we analyzed a variety of NPs of different compositions, including viruses and purified plasma EVs. After acquisition, the median scatter intensity for each EV sample was converted to size or refractive index (RI) using Mie theory.

Methods

  1. Upon startup, the instrument was primed, cleaned and flushed.
  2. Polystyrene (PS) and silica (Si) beads were diluted and titrated with HPLC water.
  3. Viruses were diluted and titrated with PBS.
  4. Extracellular Vesicles (EVs) were prepared from fresh human blood as follows:
    1. 2mL of K3-EDTA blood was first aliquotted into 12x75mm centrifuge tubes. Cells in larger volumes, further away from the max radius, do not pellet as well within a short time frame and large EVs pellet with longer time frames, so if a larger volume is needed, increasing the number of tubes works better than a greater volume per tube.
    2. The blood was centrifuged for 5 min at 200xg to pellet the majority of cells and platelets.
    3. Roughly 1mL of platelet-poor plasma (PPP) was removed from the top, careful to minimize collection of the platelet-rich plasma near the WBC layer.
    4. The PPP was filtered through a 200nm syringe filter to remove residual large particles.
    5. Finally, the filtered PPP was further purified using Izon size-exclusion chromatography (SEC) columns to remove particles smaller than 70nm.
    6. EVs were incubated with antibodies for 1 hour in the dark at RT, and then diluted 1:1K to 1:4K in PBS + 0.2% PFA prior to acquisition.
  5. All samples were acquired on a CytoFLEX-S N-V-B-R flow cytometer, and the data were analyzed in CytExpert v2.3.
  6. The median sizes of the EV fractions were analyzed by dynamic light scatter (DLS) using cumulant analyses of Brownian motion on a DelsaMax Pro. Each sample was read 10x for 10x 2-second acquisitions per read.

Materials

Materials for Characterizing the Light-Scatter Sensitivity of the CytoFLEX Flow CytometerMaterials for Characterizing the Light-Scatter Sensitivity of the CytoFLEX Flow Cytometer

Reference Standards and Viruses

Polystyrene

Reference Standards and Viruses: Polystyrene

  • 60nm PS is detected just above threshold.
  • 70nm PS is resolved.
  • 80nm PS is the official CytoFLEX spec.

Silica

Reference Standards and Viruses: Silica

  • 98.6nm Si beads are fully resolved.
  • The 98.6nm Si beads overlap with plasma EVs in both size and RI.

Viruses

Reference Standards and Viruses: Viruses

  • Adenovirus is 95nm.
  • HIV-1 is 100nm.
  • MLV is 110nm.

Mie Theory RI Curves Scaled to VSSC-H

Mie Theory RI Curves Scaled to VSSC-H

Characterization of the Light-Scatter Sensitivity for Detecting Extracellular Vesicles

CD61+ Platelet EV Sample Preparation

CD61+ Platelet EV Sample Preparation

  • CD61+ platelet EVs can be easily detected directly in whole blood or plasma.
  • To purify EV fractions, the larger particles were first removed by centrifugation and filtration, and then the smaller particles were removed by SEC, narrowing the sample range to approximately 70-200nm.

Analyses of Light Scatter vs. Size for Plasma EVs

Analyses of Light Scatter vs. Size for Plasma EVs

Analyses of Light Scatter vs. Size for Plasma EVs

Analyses of Light Scatter vs. Size for Plasma EVs

DLS Size Controls

DLS Size Controls

Discussion

Ultimately, the CytoFLEX is highly sensitive for nanoparticle detection. We found that the CytoFLEX could fully resolve 70nm PS and 100nm Si NPs, as well as 95nm Adenovirus, 100nm HIV-1, and 110nm MLV. Moreover, we were able to detect platelet EVs at least as small as 65nm in diameter using only a VSSC trigger and CD61 labeling to identify the population. Further analysis revealed that the RI of the smallest EVs increases with decreasing diameter, expanding the lower range of EVs detectable by light scatter. This effect has been observed in literature.

The CytoFLEX is for Research Use Only. The Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.

 

 

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