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Browsing Modes for Business Users
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Inspect mode – when you PunchOut to Bio-Rad from a previously created requisition but without initiating an Edit session, you will be in this mode. You cannot modify any Cart contents
Simultaneous Analysis of Bacterial Cells and Single Membrane Vesicles (MV)
Written by: Yasuma Yoshizumi, Alex Rodriguez, Richard Cuthbert
Introduction
Membrane vesicles (MVs) are membrane-bound structures released by bacteria. Interest in their biological roles has grown in recent years because they support several key processes, including cell-to-cell communication through the transfer of signaling molecules and nucleic acids (DNA/RNA), delivery of virulence factors to infected animal and plant cells, immune evasion by acting as decoys against viruses and host defenses, and nutrient cycling in soil and aquatic environments, including oceans.
Through these functions, MVs contribute to bacterial survival and growth and are important components of the broader biological network linking microorganisms with each other and with animals and plants.
Research into MVs from non-environmental bacteria has also advanced understanding of infectious disease mechanisms and supported the development of new therapeutic and preventive approaches, including modified MV-based vaccines, cancer therapies, and immune response modulation. This technical note presents an example of using the ZE5 Cell Analyzer to simultaneously measure bacterial cells and the MVs they produce and release.
Materials and Methods
Sample Preparation
Bacterial cells were grown in standard culture conditions and MVs were isolated from culture supernatants using ultracentrifugation. Following centrifugation, MVs were resuspended in PBS at the concentration stated in Table 1. Large particles were removed by filtration by passing samples through a 0.22 µm filter (Figure 1).
Table 1. Reagents used.
| Type | Sample name | Concentration |
|---|---|---|
| Buffer | PBS *0.1 µm filter-treated | |
| Sample | Bacteria-derived MV | Approx 1 x 1016 / 200 µL |
| Sample | Bacterial cells | Approx 1 x 106 / 200 µL |
Fig. 1. Sample workflow.
Sample Staining and Analysis
Following filtration, samples were stained by incubation with dyes at the concentrations listed in Table 2. In samples that included bacteria, these were re-introduced following filtration at the concentration stated in Table 1. Incubation with dyes was performed at room temperature for 15 minutes. Single-stained controls were used to set compensation, and buffer-only, stain-only, and unstained MV controls were used to ensure appropriate gating.
Following incubation, no additional washing was performed, and samples were analyzed immediately under the analysis conditions listed in Table 3. Analysis was performed using a 5-laser ZE5 Cell Analyzer equipped with a small particle detector (SPD) (12004279). Following data acquisition, data analysis was performed using Everest Software and FCS Express.
Table 2. Experiment reagents.
Reagent |
Final concentration |
Ex/Em (nm) |
Supplier |
Cat |
|---|---|---|---|---|
|
FM 4-64 Dye |
2.5 mg/mL |
500/734 |
ThermoFisher |
T3166 |
|
DiR |
250 ng/mL |
640/780 |
ThermoFisher |
D12731 |
|
Hoechst 33342 |
12.5 µg/mL |
352/461 |
Dojido Labs |
H341 |
|
Megamix-Plus SSC |
- |
495/520 |
BioCytex |
7803 |
DiR, DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide); FM 4-64 Dye, (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide); SSC, side scatter.
Table 3. Instrument run conditions.
Measurement conditions |
|
|---|---|
|
Flow rate |
0.1 μL/sec |
|
Stop volume |
20 μL |
|
Temperature (loader) |
4??? |
Results
Simultaneous Detection of MVs and Bacterial Cells
Following sample preparation and staining with FM 4-64, MVs and bacterial cells were analyzed by flow cytometry using the ZE5 Cell Analyzer. Using the small particle detector, 405 nm FSC was used to assess particle size. A clear separation could be observed between MV and bacterial cell populations. MVs were in general FM 4-64 dim with moderate FSC, whereas bacterial cells showed strong positive signal for FM 4-64 with comparatively high FSC intensity (Figure 2).
Fig. 2. Simultaneous detection of MVs and bacterial cells. Representative dot plots showing FM 4-64 fluorescence intensity against 405 nm FSC intensity, measured using the small particle detector. PBS control without cells or MVs (A), sample with MVs only (B), bacterial cells only (C), and a combined sample with both MVs and bacterial cells (D). FM 4-64, (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide); FSC, forward scatter; MV, membrane vesicles.
MV Nucleic Acid Detection
To assess MV nucleic acid content, MVs were incubated with Hoechst 33342, which is a membrane-permeable dye that binds to double-stranded DNA, as well as DiR, a lipophilic membrane stain.
DiR signal intensity showed a clear population from background, and comparison to size controls showed that the majority of MVs were between 50 nm and 240 nm in diameter (Figure 3A–C). DiR-positive staining MVs showed a clear increase in the signal intensity of Hoechst 33342, indicating that 63.3% of MVs were positive for double-stranded DNA (Figure 3D–E).
Fig. 3. Nucleic acid detection in MVs. Gating strategy showing DiR membrane staining and double-stranded DNA content as evidenced by Hoechst 33342 fluorescence. Unstained control (A) and DiR signal versus 405 nm FSC signal in stained MVs (B). Bead size control showing relative 405 nm FSC signal (C). Hoechst 33342 staining of unstained (D) and stained MVs (E). DiR, DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide); FITC, fluorescein isothiocyanate; FSC, forward scatter; MV, membrane vesicles.
Conclusions
Membrane staining enabled simultaneous detection of bacterial cells and individual membrane vesicles (MVs) using the ZE5 Cell Analyzer. Bacterial cells and MVs could be resolved using 405 nm FSC, which showed clearly distinct populations. Double staining with membrane and nucleic acid dyes also indicated that both MVs and their internal nucleic acids could be detected. Analysis showed that 63.3% of MVs contained double-stranded DNA and were between 50 nm and 240 nm in diameter.
By combining high sensitivity, high-resolution measurement, multiparameter analysis, robust software and hardware noise reduction, and high-speed acquisition, the ZE5 Cell Analyzer supports analysis of bacteria–MV interactions, MV uptake, and nucleic acid localization within MV membranes—processes that were previously difficult to study. Its temperature-controlled loader, operating from 4 to 37°C, may also support MV storage-stability studies during vaccine formulation development and related applications.
Overall, the ZE5 Cell Analyzer offers a valuable platform for analyzing both cell-sized particles and microparticles such as MVs, supporting single-particle measurement and analysis.
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