This protocol provides small organism researchers with a sensitive, quantitative, and cost efficient method for detecting the induction of nitric oxide synthase activity. First, dissect whole tissue like adult drosophila of brain, incubate the intact tissue in insect culture medium to allow for the diffusion of nitric oxide. Now, separate the culture medium and tissue sample.
Then mix the conditioned culture medium with modified grease reagent to detect the nitrite levels and indicator of nitric oxide production. Next, measure the absorption of the colored reaction product and quantify the values against a standard curve for nitrite concentration. Ultimately, this color metric assays used to show the endogenous catalytic activity of nitric oxide synthase.
While this method was designed to provide useful insight into gsof and neurobiology and innate immunity, it can also be applied to other model systems such as invertebrate models of human disease and development, as well as tissue outside of the central nervous system. Aliquot 50 microliters of grace's insect medium into the appropriate number of wells of a 96 well microtiter plate or in centrifuge tubes as demonstrated here and label suitably fully anesthetize the flies. Then transfer several flies to a microscope slide.
Add a small amount of PBS as a dissection buffer, then decapitate the fly heads under a dissection microscope, avoiding desiccation, discard the fly bodies. Now extract the full brain carefully removing cuticle, particulates and any non brainin tissue like the eye pigment tissue. Transfer the dissected brain into the appropriately labeled tube and repeat the dissections for a minimum of 20 brains per group.
Keeping the samples on ice once harvesting of a working set is complete, incubate the brain samples for six hours at 25 degrees Celsius under light shaking. Prepare the modified grease reagent as directed by the manufacturer and keep in the dark until use. Then construct a nitrite standard curve using a series of eight serial dilution of sodium nitrite.
Also label two sterile 0.5 milliliter centrifuge tubes for each treatment group as well as the control at the appropriate time points. Transfer the graces medium of each sample from the microtiter well into the corresponding micro centrifuge tube pellet any brain or tissue particulates by centrifugation at 8, 175 times gravity for two minutes without disrupting the pellet. Transfer 30 microliters of the supernatants into the second set of corresponding tubes.
Add an equal volume of grease reagent to each well after five to 10 minutes, but within 30 minutes. Measure the absorbance at 548 nanometers. Repeat each reading a minimum of three times and average three outs.
Be sure to blank the machine between every six to 10 readings and run the blank control for the determinations of standard deviation. Now to calculate the relative nitrite level in each sample. First, plot the values of the known concentrations from the nitrite standard curve using absorbence on the Y axis in concentration on the x axis.
Determine the slope of the best fit line and solve the equation for X.Now, simply substitute the absorbance values for Y and solve for X, which represents concentration. This drosophila adult male brain was dissected two to three days post eclo with midbrain and optic lobe intact. A typical paraquat toxicity curve establishes an effective lethal dose and toxic treatment range between 1.25 10 millimolar cot.
Co-treat of flies with paraquat and lna. A competitive inhibitor of nitric oxide synthase resulted in a significant rescue of lifespan. Tation caused by paraquat ingestion flies cot treated with paraquat and inactive DA isomer showed no improvement in survival.
Together these results support the hypothesis that suppressing inflammation through the inhibition of NOS enhanced survival of paraquat treated flies as further validation of a NOS mediated paraquat response. Changes in NOS protein levels were directly measured. NOS protein levels increased in a paraquat concentration dependent manner.
Treating flies with 10 millimolar paraquat over exposure durations ranging from six hours to 30 hours, results in an initial increase, then a decrease as exposure time increased. These data are consistent with the patterns of nitrite levels observed in our variant of the grease assay. Interestingly, there is a linear relationship between the increase in paraquat concentration and the magnitude of the inflammatory response as defined by the secretion and detection of nitric oxide.
Under basal levels, heterozygous punch mutants, which produce a lower amount of the NOS cofactor BH four, secrete slightly lower levels of nitric oxide. A variation that would be difficult to detect using previous detection methods such as NAD pH, diaphra HISTOCHEMICAL assay. When Fed paraquat, a pronounced increase in nitric oxide levels was observed in the punch mutant, however, significantly less than the wild type treated flies when working with toxins or chemicals.
It is vital to establish both a time of exposure curve as well as a concentration based toxicity curve since these conditions can dramatically alter the sensitivity of the assay. For example, treatment with five millimole or paraquat results in maximum NO detection after 24 hours of exposure. Comparatively exposure to 10 millimolar paraquat resulted in more rapid induction, but also more rapid decay of activity during extended exposures.
No molecules are unstable and highly reactive. A successful assay must therefore include incubation conditions that provide an optimal balance between continued induction of NOS and rapid turnover of no. In this experiment where dissected brains were incubated in the culture medium prior to adding the grease reagent, a six hour incubation at room temperature produced maximum nitrite levels in the subsequent grease reaction.
In While attempting this procedure, it's important to remember that nitric oxide is extremely unstable and undergoes rapid turnover, which could lead to loss of assay sensitivity. If incubation times are not carefully controlled and monitored, be sure to test multiple conditions in your model in order to achieve reproducible and reliable results.
