Fura-2 AM LeakRes Documentation

Fura-2 AM LeakRes Product Specifications

Cell permeable acetoxymethyl ester (AM) derivative of Fura-2 Leakage Resistant with spectral properties similar to the parent compound.

Molecular Weight

1298 g/mol

CAS#

172890-84-5

Solubility

DMSO

Kd

145 nM

Handling and Storage

Store at -20°C. Protect from light and moisture

Shelf Life

Valid for one year after delivery, if stored properly

TLC

Reverse phase C18 on silica

Solvent

5:2 Ethyl Acetate/Hexanes

Rf

0.4

HPLC

Column

C18

Detector Settings

254 nm, 371 nm

Purity

> 95%

Absorbance Spectrum

Solvent

Ethyl Acetate

Absorbance max

371 ± 3 nm

ε

33000 M-1cm-1

Fluorescence Spectrum

Solvent

Ethyl Acetate

Excitation max

369 nm ± 3 nm

Emission max

471 nm ± 3 nm

1H NMR

All relevant peaks present

Solvent

Deuterated acetone

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Fura-2 AM LeakRes Results

Fura-2 is one of the first commercial fluorescent calcium indicators introduced by Dr. Tsien and produced by Molecular Probes in 1986. It is now so common in the field that it appears in standard molecular cell biology textbooks.

Retention of Fura-2 LeakRes and Leakage of Fura-2 Over Time

Figure 1 | Figure 2

  • Images were taken at every 20 minutes. Fura-2 cells show loss of significant fluorescence by T = 40 minutes. Fura-2 LeakRes cells retain fluorescence even at T = 100 minutes.

Experimental Methods

Figure 2

BPV cells, adhered to coverslips, were loaded with Fura-2 LeakRes(AM) or Fura-2(AM) as described in Materials and Methods. Cells were mounted in a Sykes-Moore chamber and placed on a water-jacketed holder of a Zeiss IM-35 microscope. The temperature was maintained at 37°C in the sample chamber by a thermostatically controlled circulating water bath. Images were acquired with a Hammamatsu SIT camera and a Photon Technology Image Master illumination and acquisition system. Images of the same microscope field were recorded at 360 nm, excitation at 20 minute intervals beginning immediately after cells were washed. Camera gain and intensifier voltages were set based on the brightness of cells at the first time point and maintained constant thereafter. Between, the acquisition of light was blocked by a shutter. (A-F) The upper series of photographs shows the pattern of fluorescence change for Fura-2 LeakRes loaded BPV cells. The lower series of photographs (G-L) shows the corresponding changes in Fura-2 loaded BPV cells.

Figure 1

Retention of Fura-2 LeakRes and leakage of Fura-2

Fura-2 LeakRes K+ Salt Results

Note: 322 T lymphoma cells were loaded with either Fura-2 or Fura-2 LeakRes and set in calcium buffer. Leakage of Fura-2 or Fura-2 LeakRes into the exterior calcium buffer resulted in increased fluorescence overall. This fluorescence was plotted over time.

Figure 2

Decrease in fluorescence due to leakage of indicator

Fura2 LeakRes K+ Salt Results

The top row shows images of BPV cells loaded with Fura-2 LeakRes, whereas the bottom row shows BPV cells loaded with Fura-2. Images were taken at every 20 minutes. Fura-2 cells show loss of significant fluorescence by T = 40 minutes. Fura-2 LeakRes cells retain fluorescence even at T = 100 minutes.

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