Intracellular Nitric Oxide (NO) Assay Kit

Catalog No. ABIN2345133

Product Details

Reactivity: Others

Detection Method: Fluorometric

Application: Immunoassay (IA)

Brand: OxiSelect™

Sample Type: Cell Extracts, Cell Samples

Sensitivity: 3 nM

Characteristics: The OxiSelect™ Intracellular Nitric Oxide Assay Kit is a cell-based assay for rapid detection of intracellular NO or NOS activity in cultured cells. In brief, the cell-permeant NO probe passively diffuses into cells and is deacetylated by cellular esterases to a non-fluorescent intermediate. When intracellular nitric oxide encounters this intermediate, it rapidly oxidizes to a highly fluorescent, triazolo-fluorescein analog. The fluorescence intensity is proportional to the NO levels within the cell cytosol (detection limit of ~3 nM). The kit's provided Nitric Oxide Fluorometric Probe is NO specific, has excellent photostablity and pH stability, and is detected with standard fluorescein filters. The probe is suitable for flow cytometry, fluorescent microscopy, and fluorescent microplate detection. Each kit provides sufficient reagents to perform up to 96 assays, including blanks, standards, and unknown samples.

Components:

1. NOS Inhibitor, L-NNA : One vial containing 100 mg of L-NNA (CAS Number 2149-70-4, MW 219.20). L-NNA is a competitive inhibitor of nitric oxide synthase (NOS).
2. 5X Cell Lysis Buffer : Two 1.5 mL vials.
3. 500X Nitric Oxide Fluorometric Probe : One 20 μL amber vial.

Material not included:

1. Sterile DPBS containing magnesium and calcium
2. Cell culture medium
3. 37 °C incubator
4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
5. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
6. 96-well tissue culture plate Note: A tissue culture treated, 96-well fluorescence microtiter plate is required for kinetic/time course experiments.
7. 96-well fluorescence microtiter plate
8. Fluorescent microplate reader capable of reading 485 nm (excitation) and 530 nm (emission)
9. (Optional) L-Arginine Note: Found in most culture media
10. (Optional) β-NADPH, LPS, IFN-γ, NOS Inducers, NOS Inhibitors

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Direct, rapid quantitation of intracellular nitric oxide in cultured cells
• Detects nitric oxide as low as 3 nM
• Fluorometric probe is nitric oxide-specific and has excellent photostability and pH stability
• Suitable for flow cytometry, fluorescent microscopy, and fluorometric microplate detection

Reagent Preparation:

• NOS Inhibitor, L-NNA: Weigh out the NOS Inhibitor and dissolve at 0.22 mg/mL, or ~1 mM, in water (L-NNA is difficult to dissolve at concentrations above 0.5 mg/ mL). Stir to homogeneity, then sterile filter. Further dilute to the desired final concentration in media. Prepare only enough for immediate applications. Do not store diluted solutions.
• 5X Cell Lysis Buffer: Thaw the 5X Cell Lysis Buffer concentrate at room temperature. Immediately before use, dilute to 1X with deionized water. Stir to homogeneity.
• 500X Nitric Oxide Fluorometric Probe: Thaw the NO Fluorometric Probe at room temperature during assay preparation. Immediately before use, dilute the NO Fluorometric Probe to 1X concentration as follows: o If detection will be done in a fluorescence microplate reader, dilute 1:500 with either DPBS or culture media, preferably without phenol red or BSA, which may reduce fluorescence. Sensitivity of cells to serum-free environments should be considered when choosing the diluent. Briefly vortex to homogeneity. Do not store diluted solutions. o If detection will be done by fluorescence microscopy, dilute 1:500 with either DPBS or serum-free media only. Media containing serum will quench the probe's intracellular fluorescence and should not be used. Briefly vortex to homogeneity. Do not store diluted solutions. Note: For longer term storage, the 500X NO Fluorometric Probe should be aliquoted and frozen at -20 °C to avoid multiple freeze/thaws.

Assay Procedure:

I. Cell Seeding

1. Harvest and resuspend cells in culture medium at 2-5 x 105 cells/mL.
2. Seed 200 μL in each well of either a clear or black, 96-well tissue culture plate. If performing kinetic/time course experiments, a black 96-well tissue culture plate is required. Note: Overnight induction of NOS activity (e.g. β-NADPH, LPS, IFN-γ) may be performed immediately after cell plating.
3. Incubate overnight at 37 °C, 5 % CO2 (cells should be > 80 % confluent).

II. Induction of NOS Activity

1. Prepare and mix all reagents thoroughly before use.
2. Perform induction of NOS activity to desired wells. Induced, non-induced, media only, and blank wells (without NO Probe) should be assayed in triplicate. NOS inhibitors may also be added.
3. Proceed to Section III, IV or V for the desired detection method. 5

III. Kinetic/Time Course Experiments in Fluorescence Plate Reader (Black Plate Required)

1. Aspirate/remove media from the wells and add 100 μL of 1X Nitric Oxide Fluorometric Probe (see Preparation of Reagents section above) to each tested well. NOS inducers/inhibitors may also be added. Note: Media-only wells (without cells) should be included and subtracted as background.
2. Cover the plate wells to protect the reaction from light.
3. Incubate at 37 °C for the desired time period(s) (typically 30-120 minutes).
4. Read the fluorescence with a fluorometric plate reader at 480 nm/530 nm.

IV. End Point Experiments in Fluorescence Microtiter Plate Reader (Black or Clear Plate)

1. Aspirate/remove media from the wells and add 100 μL of 1X Nitric Oxide Fluorometric Probe (see Preparation of Reagents section above) to each tested well. NOS inducers/inhibitors may also be added. Note: Media-only wells (without cells) should be included and subtracted as background.
2. Cover the plate wells to protect the reaction from light.
3. Incubate at 37 °C for 2 hours.
4. Add 100 μL of 1X Cell Lysis Buffer (see Preparation of Reagents section above) to each tested well. Mix well and incubate for 5 minutes.
5. For measurement: • If already in a black 96-well tissue culture plate, wells can be directly read in a fluorometric plate reader at 480 nm/530 nm. • If in a clear 96-well tissue culture plate, transfer 150 μL of the mixture to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorometric plate reader at 480 nm/530 nm. V. Fluorescence Microscopy Detection
   1. Aspirate/remove media from the wells and discard. Gently wash the wells 2 times with 250 μL DPBS (containing magnesium and calcium). Remove the last wash and discard.
   2. Add 100 μL of 1X Nitric Oxide Fluorometric Probe (see Preparation of Reagents section above) to each tested well.
   3. Cover the plate wells to protect the reaction from light.
   4. Incubate at 37 °C for 30-120 minutes.
   5. View cells with a fluorescence microscope using FITC filter set. Note: To reduce the background fluorescence during imaging, aspirate the media/NO Fluorometric Probe and replace with fresh DPBS. 6

Restrictions: For Research Use only

Handling

Storage: -20 °C

Storage Comment: Store the entire kit at -20°C. Avoid multiple freeze/thaws by aliquoting. The Nitric Oxide Fluorometric Probe is light sensitive and should be maintained in amber tubes.

References

Hung, Castro-Lopez, Cole: "Card9- and MyD88-Mediated Gamma Interferon and Nitric Oxide Production Is Essential for Resistance to Subcutaneous Coccidioides posadasii Infection." in: Infection and immunity, Vol. 84, Issue 4, pp. 1166-75, (2016) (PubMed).

Paesano, Perotti, Buschini, Carubbi, Marmiroli, Maestri, Iannotta, Marmiroli: "Markers for toxicity to HepG2 exposed to cadmium sulphide quantum dots; damage to mitochondria." in: Toxicology, Vol. 374, pp. 18-28, (2016) (PubMed).

Nasrallah, Knezevic, Thai, Thomas, Göttgens, Lacaud, Kouskoff, Pimanda: "Endoglin potentiates nitric oxide synthesis to enhance definitive hematopoiesis." in: Biology open, Vol. 4, Issue 7, pp. 819-29, (2015) (PubMed).

Syed, Lall, Chamcheu, Haidar, Mukhtar: "Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma." in: Archives of biochemistry and biophysics, Vol. 563, pp. 108-17, (2014) (PubMed).

Target Details

Background: Reactive nitrogen species (RNS) and reactive oxygen species (ROS) work together to damage cells and have been implicated in the pathogenesis of several disease states. RNS are a family of molecules derived from nitric oxide (NO) and superoxide anion (O -2 ), produced via nitric oxide synthase (NOS) and NADPH oxidase, respectively . Nitric oxide is an established mediator in vascular diseases, diabetes, renal ischemia, atherosclerosis, inflammatory diseases, and cancer. However, because of its extremely short half life, studies of NO and its physiological role have been challenging. : Reactions leading to generation of NO and RNS. [1]