Preparation of ENU Solution
ENU is very sensitive to light, humidity, and pH. Dilute a new container of ENU prior to each weekly injection and protect from light using a foil wrap. Inject mice (Step 7) within 3 h of diluting the ENU.
Dissolving and Diluting ENU
1. In an efficient chemical fume hood, dissolve the ENU by injecting 10 mL of 95% ethanol into the ISOPAC container of ENU. Gently agitate the suspension until the ENU goes into solution. Use the warmth from your hands to warm the containment vessel.
Handlers should wear plastic gloves, lab coats, and masks.
The solution should be clear and yellow in color when the ENU is completely dissolved. This will take up to 10 min.
See Troubleshooting.
2. Dilute the ENU using one of the following methods:
i. For a standard solution, inject 90 mL of phosphate/citrate buffer into the ISOPAC container vented with an 18-gauge needle. Mix thoroughly.
ii. To prepare a more dilute solution, remove 5 mL of ENU from the ISOPAC container prior to dilution and place it directly into 50 mL of inactivating solution (e.g., 0.1 M KOH or alkaline sodium thiosulfate). Discard after a minimum of 2 h. Inject 95 mL of phosphate/citrate buffer into the ISOPAC container.
A more dilute solution is preferable for injection of small quantities of ENU per mouse.
Determining Concentration by Spectrophotometry
Because the amount of ENU per container varies, the concentration of each prepared ENU solution must be determined empirically. Measurement of concentration also controls for dilution errors.
3. Transfer 400 μL of the suspended ENU to a disposable plastic cuvette and bring the volume to 2000 μL with phosphate/citrate buffer (a 1:5 dilution). Prepare a 1:50 dilution of 95% ethanol in phosphate/citrate buffer (also in a plastic disposable cuvette) to use as a blank. Determine the OD398nm of the ENU relative to the blank.
The OD at this wavelength will vary depending upon the concentration of the ENU solution.
4. Calculate the concentration of the ENU in solution based upon the observation that a 1 mg/mL solution gives an OD398nm of 0.72.
To ensure that the spectrophotometer is working appropriately, perform a wavelength scan from 350 to 450 nm. This scan should give a peak reading at OD398. We recommend performing the scan at least once per year.