Blood collection, serum and plasma extraction, and tissue homogenate preparations were carried out as per the manufacturers’ protocol. Plasma hormone levels were measured using ELISA kits [Secretin, Angiotensin II and Vasopressin (Phoenix Pharmaceuticals Inc.), Renin LS F508 kit (Life Span BioSciences, Inc.), Aldosterone ELISA Kit (Enzo Life Science)], VEGF Quantikine ELISA Kit (R&D Systems), NO Assay Kit (Abcam) and plate reader at the respective wavelengths. Results were analyzed and compared between groups.
No assay kit
Manufactured by Abcam
Sourced in United States
The NO assay kit is a laboratory tool designed to quantify nitric oxide (NO) levels in various biological samples. It provides a simple and reliable method to measure NO concentration.
Automatically generated - may contain errors
Lab products found in correlation
Daf fm da, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Aldosterone elisa kit, by Enzo Life Sciences (1 mentions)
Vegf quantikine elisa kit, by R&D Systems (1 mentions)
Confocal microscope, by Leica camera (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Ros kit, by Abcam (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Vs0101, by Sartorius (1 mentions)
Mbs263618, by MyBioSource (1 mentions)
Elisa kit, by BioLegend (1 mentions)
Spectramax 340pc, by Molecular Devices (1 mentions)
Griess reagent, by Abcam (1 mentions)
Catalase assay kit, by Cayman Chemical (1 mentions)
Mda assay kit, by Merck Group (1 mentions)
12 protocols using no assay kit
1
Hormone and Angiogenic Factor Quantification
2
Quantifying Cell Death via Fluorescent Staining
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As previously described,[8f ] NO levels were measured using an NO assay kit (Abcam, ab65328, MA) according to the manufacturer's instructions. Cell death was measured by staining with 10 × 10−6 m Hoechst 33342 (Sigma, 14533, USA) and 5 × 10−6 m PI (DojinDo, C542, Japan) at λex = 530 nm and λem = 580 nm. Images were taken using a confocal microscope (Leica, Germany), and the percentage of PI‐positive cells was quantified with ImageJ software
3
Quantification of ROS and NO in Neutrophil-Ameba Interactions
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ROS and NO production were determined using a ROS kit (Abcam, MA, USA) [47 (link)] and a NO assay kit (Abcam, MA, USA) [48 (link)]. The production of ROS and NO was quantified from the interaction of 300,000 neutrophils with 15,000 E. histolytica trophozoites with 150 μM of kaempferol or MTZ for 90 min at 37 °C in DMEM/F12 (Sigma-Aldrich, MO, USA) + 5% SFB (Sigma-Aldrich, MO, USA) for 90 min. LPS (20 μg/mL) was used as a positive control, and trophozoites in the absence of kaempferol and trophozoites with 0.05% DMSO were used as controls. At the end of the interactions, the supernatant was removed. Samples were previously deproteinized for the nitric oxide assay. For ROS and NO determination, the manufacturer’s specifications were followed. The plates were read in a microplate reader; ROS was read at a wavelength of 480 nm excitation/530 nm and the production of NO was read at a wavelength of 540 nm.
4
Quantifying Endothelial Nitric Oxide
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Total nitric oxide (NOx) levels were determined by measuring both nitrate and nitrite in the plasma of Oasl1−/−Apoe−/− and Apoe−/− mice or in the supernatants of MAECs and HUAECs, respectively. Cells were incubated with or without TNFα and IFNγ or L-NAME for 24 h and the supernatants were collected. The plasma and supernatants were deproteinized using a 10 kDa column spin cut-off system (SARTORIUS, VS0101) and immediately assayed by the Griess method according using the NO assay kit (abcam, ab65328) according to the manufacturer’s protocol. For the analysis of the supernatant, medium alone without cells was used as the negative control.
NO production in live cells was measured using the dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA; Invitrogen, D23844), according to the manufacturer’s protocol. Briefly, for aortic tissue, mouse aortas were isolated, rapidly placed in media, split longitudinally, and pinned face up (en face preparation). Aortic tissues were pretreated with DAF-FM DA dye for 1 h, washed, and incubated in fresh media for 30 min. HUVECs were prepared and activated as indicated above. HUVECs were pretreated with DAF-FM DA for 20 min and washed for 15 min. Images were acquired by confocal microscopy (Carl Zeiss, LSM 780) with an FITC filter and the MFI was measured.
NO production in live cells was measured using the dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA; Invitrogen, D23844), according to the manufacturer’s protocol. Briefly, for aortic tissue, mouse aortas were isolated, rapidly placed in media, split longitudinally, and pinned face up (en face preparation). Aortic tissues were pretreated with DAF-FM DA dye for 1 h, washed, and incubated in fresh media for 30 min. HUVECs were prepared and activated as indicated above. HUVECs were pretreated with DAF-FM DA for 20 min and washed for 15 min. Images were acquired by confocal microscopy (Carl Zeiss, LSM 780) with an FITC filter and the MFI was measured.
5
Nitric Oxide Quantification in Brain
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NO assay kit (ab65328, Abcam Co., Waltham, MA, USA) analyzed the content of NO in brain tissue homogenate, following the manufacturer’s instructions. The activities of TNOS (MBS723386), iNOS (MBS263618), and constitutive/endothelial nitric oxide synthase (MBS160509) were measured using kits obtained from MyBioSource consistent with the manufacturer’s instructions.
6
Nitric Oxide Quantification in Neurons
7
Quantifying Inflammatory Cytokines and Nitric Oxide
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Inflammatory cytokines - TNF-α, IL-1β and IL-6 in the plasma were determined using ELISA kits (Biolegend). Levels of NO were assessed using NO assay kit (ab65328, Abcam), where utilizing nitrate reductase nitrate are converted to nitrites that gets converted to a deep purple azo compound with Griess Reagent. The intensity of the chromophore accurately reflects nitric oxide levels were measured at 540 nm using a 96-well microplate reader (Spectra MAX 340PC, Molecular Devices). The absorbance values were further analyzed using the software (Softmax Pro). The amount of NO was calculated using sodium nitrite (0–150 µM) as standard.
8
Determining Nitric Oxide Levels
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NO production was determined based on the levels of nitrate and nitrite in the culture media using an NO assay kit (fluorometric method; Abcam), according to the manufacturer's protocol (for further details, please see the Supplemental Material).
9
Quantifying Nitric Oxide in HUAECs
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To measure the total nitric oxides (NO), the HUAECs were incubated with VEGF, GSK591, or vehicle, and the supernatants were collected, followed by deproteinizing with a ten kDa column spin cut-off system (SARTORIUS, cat# VS0101). Then, the assay was performed immediately by the NO assay kit (Abcam, ab65328) with the Griess method according to the manufacturer’s protocol. To detect the NO production in living cells, the dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA; Invitrogen, cat# D23844) was used. The HUVECs were pretreated with DAF-FM DA for 15 min under indicated conditions, and the cells were washed for 10 min. The images were analyzed with a confocal microscopy system (LSM700, Zeiss), and the mean fluorescence intensity (MFI) was analyzed.
10
Cytokine and Nitric Oxide Quantification
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The inflammatory cytokine levels of TNF-α, IL-6, Il-17A, IL-1β, and IL-23 in the serum were determined by ELISA using kits from BioLegend according to the manufacturer's protocol. Levels of NO were evaluated as nitrite accumulation using Griess reagent (NO assay kit, Abcam).
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