Daf fm diacetate

Manufactured by Merck Group

Sourced in United States

DAF-FM diacetate is a fluorescent indicator used to detect and quantify nitric oxide (NO) in biological samples. It functions by reacting with NO to produce a fluorescent benzotriazole derivative that can be measured using fluorescence spectroscopy.

Automatically generated - may contain errors

Lab products found in correlation

Spectramax id3, by Molecular Devices (2 mentions) Dihydrorhodamine 123, by Thermo Fisher Scientific (1 mentions) Daf fm diacetate, by Thermo Fisher Scientific (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Dcfh2 da, by Thermo Fisher Scientific (1 mentions) Facscalibur, by BD (1 mentions) Eclipse 80i, by Nikon (1 mentions) Human umbilical vein endothelial cells (huvec), by Merck Group (1 mentions) Dm ire2 confocal microscope, by Leica camera (1 mentions)

7 protocols using daf fm diacetate

Quantification of Neutrophil RNS and ROS

Check if the same lab product or an alternative is used in the 5 most similar protocols

Production of reactive nitrogen species (RNS) by neutrophils was determined as described by the manufacturer of DAF-FM diacetate (invitrogen). Quantification of reactive oxygen species (ROS) was determined with Dihydrorhodamine 123 (invitrogen) and measured via flow cytometry according to manufacturer’s instructions.
Briefly: For measurement of reactive nitrogen species, 50 nM PMA (Sigma-Aldrich, Taufkirchen, Germany) and 5 μM DAF-FM diacetate were added to 2x10⁵ neutrophils / well and cultured at 37°C for 45 minutes. Cells were washed with PBS and incubated for additional 15 minutes at 37°C before analysis by flow cytometry.
For quantification of reactive oxygen species, 2x10⁵ cells were stimulated with 50nM PMA after a recovery time of 45min and 30 minutes later Dihydrorhodamine 123 was added at a final concentration of 2.5 μg/ml. Neutrophils were incubated for 15 minutes with Dihydrorhodamine 123 at 37°C and for 15 minutes on ice before measuring with a flow cytometer.

Franklin C., Cesko E., Hillen U., Schilling B, & Brandau S. (2015). Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease. PLoS ONE, 10(8), e0134518.

+ Open protocol

+ Expand

Measuring Oxidative Stress and Nitric Oxide in HUVECs

Check if the same lab product or an alternative is used in the 5 most similar protocols

In the present study, HUVECs were incubated with 10 µmol/ml carboxydichlorodihydrofluorescein diacetate (DCFH2-DA; Molecular Probes, Inc., Eugene, USA) at 37°C. After 15 min incubation, the increase in DCFH2 oxidation was measured using a FACSCalibur (Becton-Dickinson) (19 (link),20 (link)). The NO level in HUVECs was measured in situ using DAF-FM diacetate (Sigma-Aldrich), as previously described (21 (link)).

HE Y., LUAN Z., FU X, & XU X. (2016). Overexpression of uncoupling protein 2 inhibits the high glucose-induced apoptosis of human umbilical vein endothelial cells. International Journal of Molecular Medicine, 37(3), 631-638.

+ Open protocol

+ Expand

Quantifying ROS and NO in Human Endothelial Cells

Cited 4 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Estimates of ROS and NO production were made using 2′,7′-dichlorofluorescein diacetate and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (diaminofluorescein diacetate; DAF-FM diacetate) (Sigma), respectively, as we described.^{[31 (link)]} To determine insulin-stimulated NO production HAECs were treated with 100 nm insulin for the last 30 min of each respective treatment as we described.^{[24 (link)-26 (link),31 (link),32 (link)]}

Bharat D., Cavalcanti R.R., Petersen C., Begaye N., Cutler B.R., Costa M.M., Ramos R.K., Ferreira M.R., Li Y., Bharath L.P., Toolson E., Sebahar P., Looper R.E., Jalili T., Rajasekaran N.S., Jia Z., Symons J.D, & Babu P.V. (2017). Blueberry Metabolites Attenuate Lipotoxicity-Induced Endothelial Dysfunction. Molecular nutrition & food research, 62(2), 10.1002/mnfr.201700601.

+ Open protocol

+ Expand

Quantifying Nitric Oxide in Desiccated Roots

Check if the same lab product or an alternative is used in the 5 most similar protocols

H. rhodopensis root tips were collected at different stages of desiccation. After all soil contaminations were removed by a quick but careful cleaning using a bristle to avoid any alteration in the hydration status, intact root tip segments were collected and stained for 30 min in 5 mM DAF-FM diacetate; (Sigma, Saint Louis, MO, USA). Fluorescence of the DAF-FM NO condensate was detected using a Nikon Eclipse 80i epifluorescent microscope mounted with an FITC filter system (excitation maximum: 475 nm; emission filter: 530–543 nm). As for the negative control/basis of normalization, the unstained root tips of identical hydration status were applied. Fluorescence micrographs were taken at 40× magnification by a Spot 7.4 Slider camera (SPOT Imaging, Sterling Heights, MI, USA) and recorded at identical exposure times. For image processing and arithmetic calculations of pixel intensities, ImageJ software (V 1.8.0) was applied. Average pixel intensity of the unstained root segments was subtracted from that of the DAF-FM NO signal after baseline correction of the background noise. Data represent the mean (±SE) of n = 9.

Georgieva K., Mihailova G., Gigova L., Popova A.V., Velitchkova M., Simova-Stoilova L., Sági-Kazár M., Zelenyánszki H., Solymosi K, & Solti Á. (2023). Antioxidative Defense, Suppressed Nitric Oxide Accumulation, and Synthesis of Protective Proteins in Roots and Leaves Contribute to the Desiccation Tolerance of the Resurrection Plant Haberlea rhodopensis. Plants, 12(15), 2834.

+ Open protocol

+ Expand

Quantifying HUVEC Nitric Oxide Levels

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human umbilical vein endothelial cells (HUVEC; pooled from 3 individual donors; LONZA) were starved for 6 h, washed in PBS, and exposed to vehicle (PBS+PBr) or the cell-free-conditioned PBS at 10% or 2%, in the presence of 10 µM 4-amino-5 methylamino-2′,7′- difluorofluorescein (DAF-FM) diacetate (Sigma, D1946). DAF fluorescence intensities were measured every 5 min for a total of 60 min using a SpectraMax iD3 (Molecular Devices). All experiments were repeated at least four times per batch and were blinded.

Tarnawski L., Shavva V.S., Kort E.J., Zhuge Z., Nilsson I., Gallina A.L., Martínez-Enguita D., Heller Sahlgren B., Weiland M., Caravaca A.S., Schmidt S., Chen P., Abbas K., Wang F.H., Ahmed O., Eberhardson M., Färnert A., Weitzberg E., Gustafsson M., Kehr J., Malin S.G., Hult H., Carlström M., Jovinge S, & Olofsson P.S. (2023). Cholinergic regulation of vascular endothelial function by human ChAT+ T cells. Proceedings of the National Academy of Sciences of the United States of America, 120(14), e2212476120.

+ Open protocol

+ Expand

Intracellular Nitric Oxide Quantification in PAOEC

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

DAF-FM diacetate staining for the determination of intracellular nitric oxide bioavailability in PAOEC cells was performed as described elsewhere61 (link). Briefly, PAOECs were seeded on WillCo-dish^® glass bottom dishes (Willco Wells, Amsterdam, NL). Wild-type (siRNA-NT) and siRNA-vWF transfected cells were treated for 24 h with 100 nM AngII or saline solution (Vehicle) and then stimulated with bradykinin, as abovementioned. Unstimulated cells served as the negative control. In order to assess NO production, cells were loaded with 10 uM of the NO-sensitive fluorescence probe DAF-FM diacetate (Sigma Chemical Co, MO, USA) in the dark at 37 °C for 30 min; cells were then washed and incubated for further 15 min in fresh medium at 37 °C to allow complete de-esterification of the probe. At the end of an experiment, fluorescence was detected within 10 fields for each condition and analyzed with a Leica DM IRE 2 confocal microscope with a setting of 40x magnification. Relative fluorescence intensity per cell was quantified using ImageJ software. The experiments were performed in triplicate.

Dushpanova A., Agostini S., Ciofini E., Cabiati M., Casieri V., Matteucci M., Del Ry S., Clerico A., Berti S, & Lionetti V. (2016). Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells. Scientific Reports, 6, 30048.

+ Open protocol

+ Expand

Quantifying Nitric Oxide in HUVEC

Check if the same lab product or an alternative is used in the 5 most similar protocols

All DAF measurements were blinded. Activated (96 h) primary human T cells were incubated in PBS in the presence of PBr for 15 minutes to make conditioned PBS. Human Umbilical Venous Endothelial Cells (HUVEC; pooled from 3 individual donors; LONZA) were starved for 6 h, washed in PBS, and exposed to vehicle (PBS) or the cell-free conditioned PBS at 10% or 2%, in the presence of 4-amino-5 methylamino-2´,7´difluorofluorescein (DAF-FM) diacetate (Sigma, D1946). DAF fluorescence intensities were measured every 5 minutes for a total of 60 minutes using a SpectraMax iD3 (Molecular Devices). All experiments were repeated at least four times per batch.

Tarnawski L., Gallina A.L., Kort E.J., Shavva V.S., Zhuge Z., Martínez-Enguita D., Weiland M., Caravaca A.S., Schmidt S., Wang F., Färnert A., Weitzberg E., Gustafsson M., Eberhardson M., Hult H., Kehr J., Malin S., Carlström M., Jovinge S., & Olofsson P.S. (2021). Identification and Characterization of Human Activation-Induced ChAT⁺CD4⁺T Cells.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!