Highly responsive to pH changes

ECGreen-Endocytosis Detection is more responsive to pH changes than fluorescently labeled dextran, which has been used in the past.
Therefore, even early endosomes can be detected with high sensitivity.

　　　　　　　Change in fluorescence intensity at various pH

Fluorescence Property

λex: 386 nm, λem: 522 nm

＜Observation conditions＞
Ex : 350 ～ 410 nm, Em : 500 ～ 560 nm

Clear visualization of intracellular vesicular trafficking

Wortmannin is known to inhibit endosomal recycling and lysosomal translocation, leading to endosomal enlargement.
These changes induced by Wortmannin were confirmed by co-staining with ECGreen (green) and the following indicators.

①Eary endosome: Rab5-RFP (red)
② Recycling endosome: Fluorescent labeled Transferin (red)
③ Late endosome: Rab5-RFP (red)
④ Lysosome: Lamp1-RFP (red)

As a result, it was confirmed that ECGreen (green) co-localizes only with enlarged early endosomes and recycling endosomes (Fig. ① and ②), but not with late endosomes or Lysosomes (Fig. ③ and ④), supporting Wortmannin's effect. ECGreen can visualize changes in the intracellular vesicular trafficking system and endosome shape.

Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 – 560 nm
Early endosomes (Rab5-RFP, red): Ex. 561 nm / Em. 560 – 620 nm
Recycling endosome (Transferrin-Alexa fluor 488 conjugate, red: pseudo-color): Ex. 488 nm / Em. 500 – 550 nm
Late endosomes (Rab7-RFP, red): Ex. 561 nm / Em. 560 – 620 nm
Lysosomes (Lamp1-RFP, red): Ex. 561 nm / Em. 560 – 620 nm

(1) Prepare HeLa cells in 8 wells of μ-Slide and incubate overnight.
(2) After washing with HBSS, 200 µl of Wortmannin (final concentration: 100 nmol/l) prepared in 10% FBS-containing MEM medium was added.
(3) Incubate at 37°C for 30 minutes
(4) 200 µl of ECGreen (diluted 1,000-fold) prepared in 10% FBS-containing MEM medium without removing the supernatant
(5) Incubate at 37°C for 30 minutes
(6) Wash the cells twice with HBSS and add MEM medium containing 10% FBS.
(7) Observation with a confocal laser microscope

Experimental example: Time-lapse imaging of Endosome localization

Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 – 560 nm
Lysosome (Lysosome staining dye, red): Ex. 561 nm / Em. 600 – 700 nm

(1) HeLa cells and culture them overnight.
(2) Wash the cells once with HBSS.
(3) ECGreen (diluted 1,000-fold) prepared in 10% FBS-containing MEM medium and lysosomal staining dye (final concentration: 100 nmol/l) were added.
(4) Observe unwashed cells at each hour using a confocal laser microscope.

Visualization of EVs uptake via endocytic pathway

Mem Dye-labeled EVs are internalized via endocytosis:
HeLa cells were incubated with 10 μmol/L ECGreen for 30 min. Then, Mem Dye-Deep Red (Code: EX03) labeled EVs (quantified as 10 µg of protein) were added to HeLa cells. After 30 or 120 min incubation, the cells were washed and observed under a fluorescence microscope (Scale Bar: 10 µm).

With or without washing after staining

When observation is performed without washing after staining, the image obtained has the following characteristics

No washing after staining:

Since the dye can remain in the cell membrane, endocytosis can be observed as it changes over time.
In this case, fluorescent signals are observed on the cell membrane because an excessive amount of dye is retained in the membrane.

With washing after staining:

Residual dye in excessive amounts on the cell membrane is removed, and fluorescent signals originating from endocytosis can be observed more clearly.

Experimental example: Observation of temperature-dependent endocytosis changes using floating cells

Temperature-dependent changes in the endocytosis of Jurkat cells were visualized using ECGreen-Endocytosis Detection and PlasMem Bright Red (product code: P505).

(Scale bar: 10 µm)

Endosomes (ECGreen, green): Ex. 405 nm / Em. 500 - 560 nm
Cell membrane (PlasMem Bright Red): Ex. 561 nm / Em. 560 - 700 nm

＜Experimental procedure>
(1) Place Jurkat cell suspension (10% FBS, RPMI) in a sample tube and incubate at 4 °C or 37 °C for 30 minutes.
(2) Dilute the ECGreen solution 1000-fold using the suspension in (1).
(3) Incubate at 4 °C or 37 °C for 30 min.
(4) Wash cells twice with HBSS
(5) Add medium containing PlasMem Bright Red (100x dilution) and suspend cells.
(6) Transfer the suspension to an imaging plate and observe cells using a confocal laser microscope

Experimental Example: Observation of Endocytosis Sequence

A431 cells were stained with ECGreen (2000x dilution) for 5 minutes, washed with PBS, and observed in time-lapse under a fluorescence microscope.
The fluorescence intensity of ECGreen tended to increase around 10 minutes after the start of observation.

<Conditions of observation>
Fluorescence microscope: Olympus IX83 inverted research microscope
Filter: Semrock Excitation filter: FF01-387/11-25 Absorption filter: FF01-542/27

Imaging camera: QImaging (now TELEDYNE PHOTOMETRICS) EM-CCD camera Rolera EM-C2
                            Duration: 60 minutes, Interval: 5 minutes
This data was kindly provided by Dr. Yusuke Oba, Department of Cell Physiology, Graduate School of Medicine, Hokkaido University.