Measurement of Cyclic Guanosine Monophosphate (cGMP) in Solid Tissues using Competitive Enzyme-Linked Immunosorbent Assay (ELISA)

Dechter Laboratory researches nitric oxide metabolism. Nitric oxide stimulates cyclic GMP formation through an adaptive homeostasis cascade. We aim to learn how aging, diet, and other factors affect this cascade. Previous research focuses on nitric oxide's formation and the resulting physiological effects on neglecting the cascade connecting the two. Our protocol measures cyclic GMP, a second messenger in the cascade. Our protocol is simple, accessible, and easily adaptable to various tissue types.

[Narrator] To begin,

Place a tissue pulverizer, tweezers, and spoon on dry ice for 10 minutes. Weigh out an excess of porcine tissue to account for loss during pulverization. Transfer the tissue to the chilled pulverizer placed on dry ice. Flash freeze the tissue by pouring liquid nitrogen into the pulverizer and allow it to fully vaporize before proceeding. Then replace the lid of the pulverizer and strike it 15 times using a mallet. Now, tare a bead homogenizer tube on a balance. Using the chilled spoon and tweezers, transfer the pulverized tissue into the tube. Pipette 0.1 normal hydrochloric acid into the homogenization tube, ensuring the volume in microliters is a multiple of the tissue mass in milligrams. Then homogenize the tissue sample using a bead mill homogenizer. Centrifuge the homogenate at 17,000 g for 30 minutes, while maintaining a sample temperature of four degrees Celsius. Transfer the resulting supernatant to a microcentrifuge tube. Centrifuge again for 10 minutes, while maintaining a sample temperature of four degrees Celsius. Pipette an aliquot of the final supernatant into a microcentrifuge tube and place it on ice. Next, pipette 9.9 milliliters of ELISA buffer into a tube labeled Standard B. Pipette 100 microliters of standard stock solution into the standard B tube. Vortex thoroughly to ensure an even suspension. Transfer 1,000 microliters of the solution from Standard B to the tube labeled Standard 1. Then add 500 microliters of ELISA buffer to the tubes labeled Standard 0 and Standards 2 through 8. Vortex Standard 1. Then transfer 500 microliters of the solution into Standard 2. Then vortex Standard 2, and transfer 500 microliters of the solution into standard 3. Continue transferring 500 microliters from each previous standard into the next, vortexing thoroughly after each edition until Standard 8 is prepared. For the acetylation of the samples, add 50 microliters of four molar potassium hydroxide to a 250 microliter sample aliquot. Immediately add 12.5 microliters of acetic anhydride. Vortex the solution for 15 seconds. Then pipette 12.5 microliters of four molar potassium hydroxide and vortex again. To the tube labeled Standard 0, add 100 microliters of four molar potassium hydroxide, followed immediately by 25 microliters of acetic anhydride. After vortexing, pipette 25 microliters of four molar potassium hydroxide into the tube and vortex briefly. Repeat the potassium hydroxide and acetic anhydride additions, followed by vortexing for the remaining standards, Add a predetermined ratio of ELISA buffer and acetylated sample aliquot to a microcentrifuge tube. Vortex Standard 0. Transfer 50 microliter aliquots of Standard 0 to five wells designated as the NSB and B naught wells of the ELISA microplate. Then add 50 microliters of ELISA buffer into the two NSB wells containing Standard 0. Using a single pipette tip, add 50 microliter aliquots of Standards 1 through 8 in duplicate wells, beginning with Standard 8 and proceeding to Standard 1. Vortex each sample. Then add 50 microliters into the appropriate wells in duplicate or triplicate. Now, pipette 50 microliters of acetylcholinesterase tracer to all wells designated as NSB, B naught, standard, and sample. Add 50 microliters of antiserum to each B naught, standard, and sample well. Cover the ELISA plate and incubate at four degrees Celsius for 18 hours. After incubation, invert the ELISA plate over a waste container to dispose of the contents. Fill the wells with wash buffer using a wash bottle. Gently agitate for five seconds. Then dump the wash buffer and tap out the remaining liquid on a paper towel. Now, transfer Ellman's reagent to a reservoir. Working quickly, use a multi-channel pipette to add 200 microliters of Ellman's reagent to the blank, NSB, B naught, TA, standard, and sample wells. Add five microliters of acetylcholinesterase tracer to the TA well. Then cover the ELISA plate with Parafilm. Place it in a light protected container to incubate at room temperature with agitation for 60 to 90 minutes. Remove the Parafilm and read the optical density at 412 nanometers Cyclic GMP concentration in porcine liver in the control group was approximately 0.0202 nanomoles per gram of tissue. In the nitrate treated group, cyclic GMP concentration increased to approximately 0.0364 nanomoles per gram of tissue.