Asante Calcium Red

Asante Calcium Red emits fluorescence at long wavelengths, where there is virtually no interference from cellular autofluorescence. Exciting at 488 nm produces dual fluorescence emission at 650 and 525 nm, enabling emission ratiometry. ACR can also be excited at 540 nm to generate fluorescence emission at 650 nm, which can be used for non-ratiometric measurement.

ACR is also available in a Low Affinity (Asante Calcium Red LowAff) version and Near Membrane(Asante Calcium Red NearMem) and Leakage Resistant (Asante Calcium Red LeakRes) versions are currently under development

 

DATA

ACR in ratiometric mode

Ratiometry offers ease of calibration and enables quantitative measurement by eliminating variables such as indicator concentration, photobleaching, indicator leakage, and cell thickness. Importantly, a red-emitting indicator significantly reduces interference from cellular autofluorescence, which typically occurs at shorter wavelengths (e.g., flavin nucleotide cofactors, FMN and FAD, emit green fluorescence peaking at ~540 nm).

Until now, only two useful ratiometric Ca2+ indicators are known: Fura-2 and Indo-1. Fura -2 allows excitation ratiometry, while Indo-1 is principally used for emission ratiometry. Both these indicators require cell-damaging UV excitation expensive UV-transmitting optics.

Excitation:  488 nm

Emission: 525/650 nm

kd:  400 nM

Solubility: H20 (salt form)  DMSO (AM form)

Molecular Weight:  1000g/mol (salt)  1200 g/mol (AM)

ACR in Non-ratiometric mode

When excited at 540 nm (Figure. 4.1), ACR emits maximally at 650 nm; it shows a 50-fold increase in intensity on going from the Ca2+-free to the Ca2+-bound form. The red fluorescence of ACR is not as intense as the green fluorescence of dyes, but is useful for many applications.

Excitation:  540 nm

Emission:  650 nm

kd:  400 nM

Solubility: H20 (salt form)  DMSO (AM form)

Molecular Weight:  1000g/mol (salt)  1200 g/mol (AM)

Typical loading protocol for AM ester of ACR

(1) Prepare stock solution of 1 – 10 mM ACR (AM) in DMSO
(2) Incubate cells in 3 – 10 µM ACR (AM) in aqueous medium containing ~0.02% Pluronic F-127 (premix the requisite volume of ACR (AM) stock in DMSO with the desired volume of 15% wt/vol stock of Pluronic in DMSO before dispersal into aqueous medium)
(3) Incubate 45 – 60 minutes at room temperature
(4)Wash cells and maintain cells in fresh medium for ~40 minutes at room temperature to ensure complete intracellular hydrolysis of the AM ester (J Kao, University of Maryland)

RESULTS

Asante calcium Red emission Spectra

FIG. 4.1 Fluorescence emission traces of a titration of Asante Calcium Red (excitation at 540 nm) showing a calcium-dependent enhancement of fluorescence but not a shift of wavelength.

 

Asante Calcium Red ratiometric titration

Emission spectra of Asante Calcium Red (excitation at 488 nm) acquired at zero (Ca2+-free) and saturating (Ca2+-bound) concentrations of Ca2+. The spectral change can be used for emission ratiometry. [J. Kao, Univ of Maryland]

ACR in embryonic rat hippocampal neurons

 

Asante Calcium Red in Neurons

Figure. 4.2 Fluorescence image of cultured embryonic rat hippocampal neurons after 10 days in vitro. The cells were loaded with 3 μM Asante Calcium Red AM for 30 minutes in culture medium and then incubated in dye-free medium for 30 minutes before imaging.

 

 

Asante Calcium Red response

Figure. 4.3 Relative change in ACR fluorescence in the cell bodies of selected neurons in response to stimulation with NMDA (300 µM) in glycine-containing, Mg2+-free saline. Images were obtained every 20 seconds. Regions of interest were drawn around the somata, and mean intensities within the regions recorded as a function of time. Percent change of fluorescence at each time was calculated with respect to an average of the first five images.  

(Image, graph, and protocol provided by provided by J Marks, University of Chicago.)

 

Figure 4.4 Emission ratiometric imaging with ACR of an ATP-evoked calcium transient in a rat vagal sensory neuron. (J Kao, University of Maryland)

Fig 4.4 (ACR Neuron Images   Graphs) resized 600

Figure 4.4 Emission ratiometric imaging with ACR of an ATP-evoked calcium transient in a rat vagal sensory neuron. (J Kao, University of Maryland)

 

 

INSERT FIG. 4.5

Figure. 4.5 Emission ratiometric imaging of rat cardiac myocyte loaded with ACR and stimulated electrically. Line traces show the time courses of fluorescence intensities at 650 and 525 nm, as well as the intensity ratio. Images on the left correspond to time points marked on the lines traces. (B Hagen, J Lederer, J Kao, University of Maryland)