~ Product Features ~

High sensitivity for intracellular Fe2+ detection

Suitable for live cell imaging

Applicable for plate reader assay

 

Kit Contents: 1 tube 24μg x1; 3 tubes 24μg x3

Storage Condition: Store at 0-5 oC and protect from light

Shipping Condition: Ambient Temperature


Product Description

Iron is the most abundant transition metal element within organisms, and it participates in various physiological activities. Recently, free iron in living cells has attracted attention because its high reactivity may be related to cellular damage or death. Free iron exists in its stable redox states, namely ferrous ion (Fe2+) and ferric ion (Fe3+). In living cells, understanding the behavior of Fe2+is considered more important than understanding that of Fe3+ because of the intracellular reductive environment, metal transporters, and the water solubility of Fe2+. FerroOrange is a novel fluorescent probe that enables live-cell fluorescent imaging of intracellular Fe2+.


Ferrous ion (Fe2+) Detection

FerroOrange

Mito-FerroGreen (M489)

Localization

Intracellular Mitochondria

Fluorescent Property

λex: 543 nm, λem: 580 nmλex: 505 nm, λem: 535 nm

Instrument (filter)

Fluorescence microscope,
plate reader (Cy3)
Fluorescence microscope
(FITC、GFP)

Sample

Live cellLive Cell

The number of assays

1 tube (24 µg)
17 assays at 35 mm dish
(final concentration 1 µmol/L)
1 set (50 µg x 2)
10 assays at 35 mm dish
(final concentration 5 µmol/L)



Experimental Example

HeLa cells treated with chelator of iron 2,2'-bipyridyl (Bpy) (100 μmol/L) or Ammonium iron (II) sulfate (100 μmol/L) were prepared. The change of intracellular Fe2+in HeLa cells was detected by the FerroOrange.


<Fluorescence Microscope>

Ex/Em = 561 nm/570-620 nm, Scale bars 20 μm
Left Control
Middle Ammonium iron (II) sulfate and 2,2'-Bipyridyl (Bpy) treated
Right Ammonium iron (II) sulfate treated
The fluorescence intensity of FerroOrange was increased in HeLa cells treated with Ammonium iron (II) sulfate compared with the findings in untreated cells; conversely, its fluorescence intensity was decreased in cells treated with Bpy.


<Plate Reader Assay>

Ex/Em = 543 nm/ 580 nm
Left Control
Middle Ammonium iron (II) sulfate and 2,2'-Bipyridyl (Bpy) treated
Right Ammonium iron (II) sulfate treated
The change of intracellular Fe2+in HeLa cells was quantified by the FerroOrange.


<Metal Ion Selectivity>

Ex/Em = 543 nm/ 580 nm

2 μL of 1 mmol/L FerroOrange and 2 μL of 10 mmol/L from each metal were added to 1mL of 50 mmol/L HEPES Buffer (pH7.4). The fluorescence intensity was measured after the reaction, for 1hr, at room temperature.


<Excitation and Emission Spectra>


Co-staining with Each Organelle Dye Reagent

FerroOrange was co-stained with each organelle’s dye reagents.
HeLa cells were stained with organelle’s dye and washed.
Then, FerroOrange was added to the cells and cells were observed under the fluorescent microscope.


Co-staining with ER Staining Dye

<Detection Condition>
FerroOrange: Ex. 561 nm, Em. 570-620 nm
ER Tracker Green (ER Dye): Ex. 488 nm, Em. 510-555 nm
Scale bars: 10 µm


Co-staining with Mitochondrial staining Dye

<Detection Condition>
FerroOrange: Ex. 561 nm, Em. 570-620 nm
MitoBright Deep Red (Mitochondrial Dye): Ex. 640 nm, Em. 650-700 nm
Scale bars: 10 µm


Co-staining with Golgi Complex Staining Dye

<Detection Condition>
FerroOrange: Ex. 561 nm, Em. 570-620 nm
BODIPY FL (Golgi Complex Staining Dye): Ex. 488 nm, Em. 510-555 nm
Scale bars: 10 µm






Q. Are there any tips for successful assay?

A. 
  1. Please do not change the media after adding the FerroOrange. By changing the media, the FerroOrange dye can leak out of cells.
  2. For data reliability, we recommend preparing Bpy(2,2‘-bipyridine) or ammonium iron sulfate (II) treated cells as the control for comparison with FerroOrange data.
  3. If the cell samples have difficulty in staining, please increase the concentration of FerroOrange working solution 1μmol/L higher than the recommended concentration. We recommend 1-5μmol/L.


Q. How can I use FerroOrange with a plate reader?

A. Please refer to the following experimental example:

<Sample>
A: No dye added (HeLa cells only)
B: Bpy(2,2‘-bipyridine) treated HeLa cells
C: Ammonium iron sulfate (II) treated HeLa cells

<Method>
  1. Add 100μL HeLa cell suspension to each well of 96 well plate (black with clear bottom) making the final concentration 10,000 cells/well. Incubate overnight in the 5% CO2 at 37 ℃.
  2. Wash Sample C with 100 μL MEM (no FBS) media three times
  3. Add 100 μL ammonium iron sulfate (II)/MEM (no FBS) solution (final concentration: 100 μmol/L) to Sample C’s wells. Incubate for 30 minutes in the 5% CO2 at 37 ℃.
  4. Wash cells with 100 μL HBSS three times
  5. Add 100 μL of 1 μmol/L FerroOrange working solution to Sample A and Sample C’s wells. Add 100 μL of HBSS solution containing FerroOrange (final concentration: 1 μmol/L) and Bpy (final concentration: 100 μmol/L) to Sample B’s wells. Incubate for 30 minutes in the 5% CO2 at 37 ℃.
  6. Measure the fluorescent intensity (Ex. 543 nm, Em. 580nm) using a fluorescence microplate reader.


Q. What is the recommended filter?

A. Excitation: 530-565 nm; Emission: 570-620 nm













Item # Description/Size Availability Qty Break Price Quantity
F374-10
24µg x1
1-2 business days 1 $140.00
F374-12
24µg x3
1-2 business days 1 $320.00

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