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Protonex™ Green 500-Zymosan A Conjugate
Product key features
The Protonex™ Green 500–Zymosan A Conjugate provides a ready-to-use, pH-sensitive fluorescent tool for monitoring phagocytosis and intracellular acidification processes in live cells.
- pH-activated fluorescence: Non-fluorescent at neutral pH and becomes strongly fluorescent in acidic compartments such as phagosomes and phagolysosomes.
- Zymosan-based targeting: Derived from yeast cell walls, Zymosan A is a potent biological substrate for phagocytic uptake.
- Multipurpose reagent: Suitable for incorporation into custom assay workflows for microscopy, flow cytometry, or plate-based readouts.
Product description
The Protonex™ Green 500-Zymosan A Conjugate is a ready-to-use reagent designed to study phagocytosis and phagosome acidification in live cells. This conjugate combines Zymosan A particles, a biologically active yeast cell wall component with Protonex™ Green 500, a novel pH-sensitive fluorophore that remains non-fluorescent at neutral pH and becomes highly fluorescent upon entering acidic environments such as maturing phagosomes and phagolysosomes.
As a standalone reagent, it enables users to integrate phagocytic detection into their own custom assays. The FITC-like excitation/emission properties of Protonex™ Green 500 make it compatible with a wide range of fluorescence imaging and detection systems. These conjugates can be used in combination with red fluorescent dyes like RFP, Calbryte™ 630 calcium dye, calcein red, or Cy5-labeled antibodies for multiplexed cell functional analysis. It is ideal for immunological research, drug discovery, and mechanistic studies of innate immune function, autophagy, or particle uptake.
Example protocol
AT A GLANCE
- Plate the cells.
- Treat cells with test compounds.
- Add Protonex Dye Zymosan A conjugates in medium.
- Incubate at 37°C for 60 minutes.
- Monitor fluorescence by microscope or fluorescence plate reader.
CELL PREPARATION
For guidelines on cell sample preparation, please visit:
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
- Plate cells overnight in a growth medium at 20,000-50,000 cells/well/100 µL in a 96-well plate.
Note: For RAW 264.7 cells used in this assay, we recommend plating 50,000 cells per well in 100 µL of medium in a 96-well plate and incubating them overnight. It is important to optimize the cell density for each cell line individually.
Note: Higher background fluorescence levels may be seen with poly-D-lysine coated microplates.
SAMPLE EXPERIMENTAL PROTOCOL
Add phagocytosis inhibitor or inducer (e.g., Cytochalasin D or LPS) at the desired concentrations. You may need to add vehicle controls to untreated wells. (For example: 11X working solution can be prepared in PBS, and 10 µL can be added to each well.)
Note: The time and concentration of phagocytosis effectors varies with cell types.
- Add the suspension of Zymosan A conjugate to the cell culture microplate in a 1:10 dilution, or 10 μL of particles added to 100 μL of cell culture medium, and mix well.
- Place the cells at 37°C for 60 minutes to 3 hours.
- Wash the cells 2-3 times with HHBS Buffer (AAT Cat# 20011) or buffer of your choice.
- Add 100 µL HHBS Buffer to each well.
- Observe plate with a fluorescence microscope using the following filter set or read plate in a fluorescence plate reader with bottom read mode.
Spectrum
References
Authors: Liu, Wei and Liu, Bo
Journal: Methods in molecular biology (Clifton, N.J.) (2025): 171-175
Authors: Agina, Onyinyechukwu Ada and Shaari, Mohd Rosly and Isa, Nur Mahiza Md and Ajat, Mohd Mokrish Mohd and Zamri-Saad, Mohd and Samad, Mohd Jamil and Hamzah, Hazilawati
Journal: Research in veterinary science (2023): 105073
Authors: Piszczek, P and Wojcik-Piotrowicz, K and Nowak, B and Guzdek, P and Novak, P and Pytko-Polonczyk, J and Gil, K and Kaszuba-Zwoinska, J
Journal: Journal of physiology and pharmacology : an official journal of the Polish Physiological Society (2023)
Authors: Pavlova, Ekaterina and Shaposhnikova, Daria and Petrichuk, Svetlana and Radygina, Tatiana and Erokhina, Maria
Journal: Methods in molecular biology (Clifton, N.J.) (2023): 203-215
Authors: Tram, Trinh T B and Ha, Vu T N and Thu, Do D A and Dinh, Tran D and Vijay, Srinivasan and Hai, Hoang T and Hanh, Nguyen T and Phu, Nguyen H and Thwaites, Guy E and Thuong, Nguyen T T
Journal: Biology of the cell (2019): 262-270
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