I can propose some methods:

1)Glucose + ATP -(hexokinase)-> glucose-6-phosphate + ADP

2)glucose-6-phosphate + NADP+ -(glucose-6-phosphate dehydrogenase)-> 6-phosphogluconate + NADPH (absorbance at 340 nm)

=originally is a method for detection glucose

1)Glycerol + ATP -(glycerokinase)->glycerol-3-phosphate + ADP

2)glycerol-3-phosphate + O2 -(glycerolphosphate oxidase)-> phosphodioxyacetone + H2O2

3) H2O2 + TetraMethylPhenyleneDiamine(or other reductant) -(horseradish peroxidase)-> H2O + TMPD*+ (abs at 610 nm)

=originally for detection triglycerides

1)ATP+NADH -(NADH kinase)-> ADP + NADPH (absorbance at 340 nm)

All of this methods must be first calibrated on ATP concentration to define usability in your concentrations of ATP.

Moreover, some fluorimeters can also works in luminometer mode, check does yours?

I have always measured ATP from microdialysis samples by luminescence, utilizing ATPLite-M kit (Perkin-Elmer, Life and Analytical Sciences). I don’t know a method to determine ATP by spectrophotometer.

I am very sorry that I can not help you.

We are also planning to do the same by ATPlite, For that we need to have a luminometer...spectrophotometer or fluorimeter are not suitable for that...am I right?

yes, you need luminometer for ATPlite(btw, I was using ATP kit by SIGMA http://www.sigmaaldrich.com/catalog/product/sigma/flaa?lang=en®ion=SX, I find it more sensitive than ATPlite), but if you use this ATP Assay kit from Abcam: http://www.abcam.com/ATP-Assay-Kit-ColorimetricFluorometric-ab83355.html you don't need luminometer...however, I haven't used Abcam kit myself, so can't really say how reliable it is. Hope this helps. Good luck

Try http://probes.invitrogen.com/media/pis/mp06645.pdf

Thank you very much Alessia Melani, Ivana Gadjanski, Ricardo Bernal for your support.

I measured ATP from lymphocytes in peripheral blood by luminescence, utilizing ATP Kit by SIGMA.

Aurel

Thank you...Aurelian Udristioiu.

I can propose some methods:

1)Glucose + ATP -(hexokinase)-> glucose-6-phosphate + ADP

2)glucose-6-phosphate + NADP+ -(glucose-6-phosphate dehydrogenase)-> 6-phosphogluconate + NADPH (absorbance at 340 nm)

=originally is a method for detection glucose

1)Glycerol + ATP -(glycerokinase)->glycerol-3-phosphate + ADP

2)glycerol-3-phosphate + O2 -(glycerolphosphate oxidase)-> phosphodioxyacetone + H2O2

3) H2O2 + TetraMethylPhenyleneDiamine(or other reductant) -(horseradish peroxidase)-> H2O + TMPD*+ (abs at 610 nm)

=originally for detection triglycerides

1)ATP+NADH -(NADH kinase)-> ADP + NADPH (absorbance at 340 nm)

All of this methods must be first calibrated on ATP concentration to define usability in your concentrations of ATP.

Moreover, some fluorimeters can also works in luminometer mode, check does yours?

The hexokinase + glucose-6-phosphate dehydrogenase coupled reaction (shown in Vladimir's message) is a reasonably standard procedure for ATP. The NADPH produced is very easy to measure at 340 nm and quite sensitive. Regardless of the method, it is necessary to calibrate with an ATP concentration series.

I remembered this too: http://www.biovision.com/atp-colorimetric-fluorometric-assay-kit-2856.html

The kit uses the reaction 1) from Vladimir's reply...I haven't used it myself, but it seems good. If you decide to use this, could you, please, give feedback here how it worked for you? Thanks

I'll surely do so...Thank you all for your support...

Good, I loved to see all suggestions. It seems nice. However I did not dosed ATP yet!

Good look!

Maria Elena.

If the detector in the nanodrop UV/VIS is sufficiently sensitive, you could turn off the light source and measure a luminescent signal. However, it needs to be calibrated. Of course, you will have to prevent external light to interfere with the phototube signal.

I have also used a scintillation counter with the detectors taken out of the normal coincidence measurement mode.

Can anyone provide references for vladimir's last method??

The main challenge with mouse tissues is a sample a preparation step, not the final detection method. You need to rapidly freeze it in liquid nitrogen (and keep frozen while homogenizing with perchloric acid or similar) to avoid ATP degradation which occurs very fast and easily. More details and references are in this discussion:

https://www.researchgate.net/post/Which_is_a_good_Tissue_Cell_Lysis_Buffer_for_Luminescent_ATP_assay