AcidiFluor™ Series
[Acidic pH detecting probe]
570-590 nm：Orange
AcidiFluor ORANGE-Zymosan A is the conjugate of AcidiFluor ORANGE-NHS and Zymosan A, a reagent commonly used for analyzing phagocytosis. By treating phagocytes with a suspension of this product, fluorescent imaging of phagocytosis is possible.
As AcidiFluor ORANGE-NHS, this product also high pH-responsivity.
| Code No. | Product Name | Size | Merck CAT No. |
Merck ( Millipore / Sigma Aldrich ) Product Name |
|---|---|---|---|---|
| GC305 | AcidiFluor™ ORANGE-Zymosan A | 1 mg |
AcidiFluor™ ORANGE-Zymosan A
Product Information
| Product Name |
target | reaction | pKa |
Absmax (nm) | FLmax (nm) | ε | Φ |
| AcidiFluor ORANGE-NHS | pH | reversible | 5.3, 6.8 | 544 | 565 | 80,000 | 0.7 |
AcidiFluor™ ORANGE-Zymosan A
Cell imaging example using AcidiFluor ORANGE-NHS and AcidiFluor-ORANGE Zymosan A
Anti-EGFR antibody was labeled with AcidiFluor ORANGE-NHS, and unreacted dye was removed by ultrafiltration. A431 cell, EGFR overexpressing cell line, was treated with purified AcidiFluor ORANGE labeled anti-EGFR antibody, then time-lapse observation was performed by using confocal microscope. After 120 min of the addition, fluorescence signal derived from AcidiFluor ORANGE began to be detected, and further 60 min later, strong fluorescence was observed. This result indicates that AcidiFluor ORANGE labeled anti-EGFR antibody was incorporated into the cells by endocytosis and then the endocytotic vesicle was acidified.
After addition of AcidiFluor ORANGE-Zymosan A to RAW264.7 cells, the increase of fluorescence intensity was observed, indicating an acidification of folliculi.
AcidiFluor™ ORANGE-Zymosan A
Detection of phagocytosis using AcidiFluor ORANGE-Zymosan A with a flow cytometer
Zymosan A is a compound prepared from yeast cell walls. It is often used to induce inflammatory responses, such as phagocytosis. AcidiFluor ORANGE-Zymosan A is Zymosan A labeled with AcidiFluor ORANGE, an acidic pH indicator. Its fluorescence intensity is highest in an acidic environment, such as phagosomes. AcidiFluor ORANGE-Zymosan A is a useful probe to measure phagocytosis, since the increase in fluorescence intensity only occurs after phagocytosis. Here we present an example data to evaluate phagocytosis of mouse macrophage-derived RAW 264.3 cells using AcidiFluor ORANGE-Zymosan A, and comparison with other products.
Fig. 1. Phagocytotic activity of RAW264.7 cells upon the concentration of AcidiFluor ORANGE-Zymosan A (AFO-Zymosan A). Each concentration of AcidiFluor ORANGE-Zymosan A was added to RAW264.7 cells, then incubated at 37°C, 5% CO2 for 1 hour. Cells were rinsed with PBS and analyzed. Left shows the summarized result, whereas the right shows the fluorescence intensity distribution in each condition. The number of fluorescently positive cells were determined as the number of cells which fluorescence intensity is above 6 × 102. AcidiFluor ORANGE was excited with a 488-nm laser, and the fluorescence intensity was measured with a 586/42 nm filter for phycoerythrin.
Protocol
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Fig. 2 Effect of cytochalasin D on phagocytosis examined using AcidiFluor ORANGE-Zymosan A. After pretreatment of cytochalasin D (phagocytosis inhibitor) at various concentrations (0 µM: red, 1 µM: yellow, 2 µM: green, 5 µM: blue, and 10 µM: purple), AcidiFluor ORANGE-Zymosan A of 50 µg/mL was added to RAW264.7 cells and incubated at 37°C, 5% CO2 for 1 hour. The cells rinsed with PBS were analyzed using a flow cytometer.Left indicates the summarized result, while the right indicates the distribution of fluorescence intensity in each condition. The number of fluorescence-positive cells indicated in the right plot are determined by the number of cells which fluorescence intensity is above 6 × 102. The inhibition ratio of phagocytosis (ri) was calculated as follows:
where R+ represents the percentage of phagocytotic cells treated with cytochalasin D, and R- represents the percentage of phagocytotic cells without the addition of cytochalasin D. AcidiFluor ORANGE was excited with a 488-nm laser, and the fluorescence intensity was measured with 586/42 nm filter for Phycoerythrin.
These data indicate that the fluorescence intensity profile well reflects the efficiency of phagocytosis by macrophages.
Bright fluorescence of AcidiFluor ORANGE-Zymosan A, in addition to its high signal-to-noise ratio, enables easier discrimination of phagocytotic cells from non-phagocytotic cells. It gives higher signal intensity compared with other pH indicating fluorophores, such as pHrodo Red and pHrodo Green.
Fig. 3 Comparison with pHrodo Red-Zymosan A and pHrodo Green-Zymosan A. Various concentrations (10, 30, 50, 100 µg/mL) of AcidiFluor ORANGE-Zymosan A, pHrodo Red-Zymosan A, and pHrodo Green-Zymosan A were added to RAW264.7 cells and incubated at 37°C, 5% CO2 for 1 hour. The cells rinsed with PBS were analyzed using flow cytometry. Left indicates the ratio of phagocytotic cells in each concentration of Zymosan A labeled with each fluorophores. Right indicates a representative distribution of fluorescence intensity measured at the concentration of 50 µg/mL. Fluorescence intensity above 3 × 102 units are determined as fluorescence positive cells. AcidiFluor ORANGE, pHrodo Red, and pHrodo Green were excited with a 488-nm laser. The fluorescence intensities of AcidiFluor ORANGE and pHrodo Red were measured with a 586/42 nm filter for Phycoerythrin, while that of pHrodo Green was measured with 537/32 nm filter for FITC. The negative control (a non-treated sample) is indicated as black broken lines.
As shown in Fig. 3, the ratio between the phagocytotic cells and the non-phagocytotic cells was the highest for AcidiFluor ORANGE-Zymosan A (left) at every concentration. In addition, AcidiFluor ORANGE-Zymosan A could better separate the phagocytotic cells from non-phagocytotic cells in fluorescence intensity distribution when compared with pHrodo Red-Zymosan A (right).
No, basically, it cannot be applied to fixed cells. Acidic pH of lysosomes and endosomes are kept by the activity of living cells and fixed (dead) cells do not keep the acidic pH.
On the other hand it could be possible to detect localization of the probes by soaking fixed cells into an acidic buffer, because localization of AcidiFluor ORANGE, which has a structure to be localized in lysosomes, antibody-labeled AcidiFuor ORANGE-NHS and HaloTag labeled HaloTag AcidiFluor ORANGE ligand can be kept for a while after fixation.
Please also refer the frequently asked questions on fluorescent probes
D. Asanuma, Y. Takaoka, S. Namiki, K. Takikawa, M. Kamiya, T. Nagano, Y. Urano, K. Hirose (2014)
Angew. Chem. Int. Ed. Engl. 53: 6085-6089 DOI:10.1002/anie.201402030
M. Isa, D. Asanuma, S. Namiki, K. Kumagai, H. Kojima, T. Okabe, T. Nagano, K. Hirose (2014)
ACS. Chem. Biol. 9: 2237-2241 DOI:10.1021/cb500654q
R. Watanabe, N. Soga, D. Fujita, K. V. Tabata, L. Yamauchi, S. H. Kim, D. Asanuma, M. Kamiya, Y. Urano, H. Suga, H. Noji (2014)
Nat. Commun. 5, Article number: 4519 DOI:10.1038/ncomms5519
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