BioxML Yellow
BioxML Yellow is a yellow fluorescent dye developed by Bioxol Ltd. specifically for the reliable and selective labeling of extracellular vesicles (EVs) as well as cellular membranes, complementing the BioxML Red dye family. The dye selectively incorporates into lipid bilayers and, upon excitation with light of an appropriate wavelength, emits a bright yellow fluorescent signal. It is suitable for flow cytometry, confocal microscopy, and nanoparticle tracking analysis (NTA) applications.
The dye is already actively used in experimental studies by the Institute of Genetics, Cell and Immunobiology at Semmelweis University.
Technical Parameters
Parameter — Specification
Excitation maximum — 550 nm
Emission maximum — 570 nm
Recommended concentration — 1–50 nM
Packaging — Lyophilized powder
Solubility — DMSO
Compatible with — FACS, confocal microscopy, NTA, live-cell and fixed-cell imaging
Storage conditions — −20°C, protected from light
Validated on the following cell lines and model systems: — A549, HEK293, H9c2, THP-1, Jurkat, COLO205, MIA PaCa-2, and DOPC liposomes.
Challenges Solved by BioxML Yellow
BioxML Yellow eliminates several common challenges in extracellular vesicle research that often compromise detection sensitivity and experimental reproducibility:
• Non-specific staining
The dye fluoresces only after incorporation into phospholipid bilayers, preventing false-positive detection of non-vesicular particles such as protein aggregates.
• Background fluorescence caused by aggregation
Thanks to its self-quenching mechanism, free, unbound dye molecules produce negligible fluorescence, eliminating disturbing background signals.
• Lengthy or sensitive antibody-based staining protocols
BioxML Yellow can replace or complement complex antibody-based workflows with a fast, direct membrane-labeling approach.
• Lack of known EV surface markers
No specific protein markers are required, as the dye labels all lipid membrane-containing vesicles.
• Weak fluorescence due to nanometer-sized EVs
Its high quantum efficiency and membrane selectivity enable reliable discrimination of small EVs—including particles below 100 nm—from background noise during flow cytometric analysis.
What BioxML Yellow Provides
• Membrane-selective localization
Ensures labeling of the entire EV population present in the sample, enabling comprehensive and accurate quantitative analysis in both NTA and flow cytometry.
• Self-quenching capability
Free dye fluorescence is automatically suppressed, allowing measurements with exceptionally low background noise and high signal-to-noise ratio, even without washing steps.
• Fast and simple workflow
Requires minimal incubation time, significantly reducing and standardizing EV detection workflows in both flow cytometry and NTA applications.
• Optimized yellow/orange emission
Specifically optimized for excitation with 532 nm and 561 nm lasers, enabling detection in PE and mCherry channels, and supporting multiplex fluorescent labeling experiments.
Protocol for EV Detection by Flow Cytometry
Dilute purified EV samples isolated from HEK293 conditioned medium 10×, then prepare a final 50× dilution for the experiment.
Prepare a stock solution of BioxML Yellow (5 mM) and dilute 25× in DMSO under sterile conditions.
Dilute the solution further 100× in PBS.
Add the dye to samples at a final 100× dilution (5 µL), resulting in a final dye concentration of 20 nM.
Based on protocol optimization studies, no additional washing steps or incubation time are required.
Notes
The PBS solution used in experiments was prepared by mixing 5 mL of 10× PBS with 45 mL Milli-Q water (Merck), followed by filtration through a 0.2 µm filter (Millipore) to eliminate physical contaminants.
Successful EV staining was confirmed using detergent lysis controls at appropriate concentrations.
Scientific Publications
Several scientific publications are already available demonstrating the application of BioxML Yellow:
Benke et al. (2023)
"MiR-200b categorizes patients into pancreas cystic lesion subgroups with different malignant potential"
Scientific Reports, 13(1), 19820.Lenzinger et al. (2025)
"Stress-induced switch in small extracellular vesicle secretion: From constitutive ‘torn bag mechanism’ to exocytosis"
bioRxiv.