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Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cells in vitro.

Räthel TR - Biol Proced Online (2003)

Bottom Line: Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells in vitro.The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated.Here we focus in addition to the previous publication (Leikert et al., FEBS Lett 2001, 506:131-134) on aspects of validation procedures as well as limitations and pitfalls of this method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacy, Center of Drug Research, University of Munich. Butenandtstr. 5-13, D-81377 Munich. Germany.

ABSTRACT
Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells in vitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 micro M) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikert et al., FEBS Lett 2001, 506:131-134) on aspects of validation procedures as well as limitations and pitfalls of this method.

No MeSH data available.


The difference in measured fluorescence intensity of DAF-2 alone or after reaction with NO released from endothelial cells becomes more significant with lower DAF-2 concentrations. Blank vials (white bars) or EA.hy 926 cells (black bars) were incubated with PBS supplemented with 100 µM of L-arginine for 5 min at 37°C in the dark. Then DAF-2 at the indicated concentrations and the calcium ionophore A23187 (1 µM) were added. 5 min later. The fluorescence of the supernatants was measured as described in Materials and Methods. All data are mean ± S.D. (n = 2 in triplicate). Differences between means were analyzed using Student’s t-test. *P< 0.05; **P< 0.01.
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Figure 1: The difference in measured fluorescence intensity of DAF-2 alone or after reaction with NO released from endothelial cells becomes more significant with lower DAF-2 concentrations. Blank vials (white bars) or EA.hy 926 cells (black bars) were incubated with PBS supplemented with 100 µM of L-arginine for 5 min at 37°C in the dark. Then DAF-2 at the indicated concentrations and the calcium ionophore A23187 (1 µM) were added. 5 min later. The fluorescence of the supernatants was measured as described in Materials and Methods. All data are mean ± S.D. (n = 2 in triplicate). Differences between means were analyzed using Student’s t-test. *P< 0.05; **P< 0.01.

Mentions: These theoretic considerations were demonstrated experimentally, as shown in Figure 1: We employed increasing concentrations of DAF-2 (0.01-5 µM in PBS) to either a blank vial (no NO source) or to A23187-activated human endothelial EA.hy926 cells (low-output NO source) followed by fluorescence measurement (λex 495 nm, λem 515 nm). Figure 1 shows that the fluorescence intensity obtained from DAF-2/DAF-2T in the supernatants of NO-producing endothelial cells does not exceed significantly the level of the fluorescence intensity obtained from DAF-2 auto-flourescence (no NO source) when concentrations of DAF-2 > 1 µM were employed. This suggests that concentrations < 1 µM DAF-2 should be employed measuring NO released from cNOS systems.


Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cells in vitro.

Räthel TR - Biol Proced Online (2003)

The difference in measured fluorescence intensity of DAF-2 alone or after reaction with NO released from endothelial cells becomes more significant with lower DAF-2 concentrations. Blank vials (white bars) or EA.hy 926 cells (black bars) were incubated with PBS supplemented with 100 µM of L-arginine for 5 min at 37°C in the dark. Then DAF-2 at the indicated concentrations and the calcium ionophore A23187 (1 µM) were added. 5 min later. The fluorescence of the supernatants was measured as described in Materials and Methods. All data are mean ± S.D. (n = 2 in triplicate). Differences between means were analyzed using Student’s t-test. *P< 0.05; **P< 0.01.
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Related In: Results  -  Collection

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Figure 1: The difference in measured fluorescence intensity of DAF-2 alone or after reaction with NO released from endothelial cells becomes more significant with lower DAF-2 concentrations. Blank vials (white bars) or EA.hy 926 cells (black bars) were incubated with PBS supplemented with 100 µM of L-arginine for 5 min at 37°C in the dark. Then DAF-2 at the indicated concentrations and the calcium ionophore A23187 (1 µM) were added. 5 min later. The fluorescence of the supernatants was measured as described in Materials and Methods. All data are mean ± S.D. (n = 2 in triplicate). Differences between means were analyzed using Student’s t-test. *P< 0.05; **P< 0.01.
Mentions: These theoretic considerations were demonstrated experimentally, as shown in Figure 1: We employed increasing concentrations of DAF-2 (0.01-5 µM in PBS) to either a blank vial (no NO source) or to A23187-activated human endothelial EA.hy926 cells (low-output NO source) followed by fluorescence measurement (λex 495 nm, λem 515 nm). Figure 1 shows that the fluorescence intensity obtained from DAF-2/DAF-2T in the supernatants of NO-producing endothelial cells does not exceed significantly the level of the fluorescence intensity obtained from DAF-2 auto-flourescence (no NO source) when concentrations of DAF-2 > 1 µM were employed. This suggests that concentrations < 1 µM DAF-2 should be employed measuring NO released from cNOS systems.

Bottom Line: Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells in vitro.The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated.Here we focus in addition to the previous publication (Leikert et al., FEBS Lett 2001, 506:131-134) on aspects of validation procedures as well as limitations and pitfalls of this method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacy, Center of Drug Research, University of Munich. Butenandtstr. 5-13, D-81377 Munich. Germany.

ABSTRACT
Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells in vitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 micro M) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikert et al., FEBS Lett 2001, 506:131-134) on aspects of validation procedures as well as limitations and pitfalls of this method.

No MeSH data available.