Assays for Qualitative and Quantitative Assessment of Mouse Urine Glucose Concentrations
Transcrição
Begin by placing a piece of clean parafilm on a flat surface. Gently pick up the mouse by the tail and set it on the parafilm.
Restrain the mouse by the scruff using the thumb and index finger, causing the mouse to urinate as a stress response. Allow for a drop of urine to fall onto the parafilm surface and then return the mouse to its cage.
Dip the reagent end of a glucose detection strip in the fresh urine drop. Immediately remove the strip and lay it flat on a surface with the color side up. Then, wait at least 30 seconds for a color to fully develop on the strip. Compare the developed color with the reference chart provided by the manufacturer.
Assemble the metabolic cage according to the manufacturer's instructions, and fill the feed assembly with powdered chow, then, attach it to the appropriate metal tab on the upper chamber.
Secure the water bottle support to the remaining metal tab on the upper chamber. Fill the empty water bottle and slide it into the bottle support, making sure the sipper tube is in the hole.
Place a single mouse in the metabolic cage and cover it with the lid, ensuring that the cage lid is fitted properly. Position the urine collection tube and feces collection tube in their respective spots in the lower chamber floor.
After 24 hours, retrieve the urine collection tube and perform a glucose assay to determine the urine glucose concentration. If the glucose assay will not be performed on the same day, freeze the urine sample in a minus 80-degree Celsius freezer.
If using a frozen urine sample, thaw it first. Prepare eight standards ranging from 25 to 0 milligrams per deciliter, according to the assay kit directions.
Prepare standard wells and urine sample wells in a 96-well plate, all in duplicates, according to the kit manual. Initiate the reaction by adding the recommended amount of enzyme mixture to all the wells. Cover the plate, and let it incubate for 10 minutes at 37 degrees Celsius. Then, read the absorbance at 500 to 520 nanometers using a plate reader.
Analyze the data according to the assay kit instructions and plot the absorbance of each standard versus the glucose concentration. Utilize this graph to determine the glucose concentration of the 24-hour urine sample. If the concentration of the urine is out of the standard range, dilute the urine sample.
