A simple method for short-term maintenance of neonatal mice without foster mothers
2Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68583, USA
3School of Biological Sciences, University of Nebraska, Lincoln, NE 68583, USA
Transferring of neonatal mice
1.Sanitize a biosafety cabinet (BSC) with Clidox for working surface and 70% ethanol for side walls.
2.Remove a cage from the rack and spray with 70% Ethanol to sanitize cage outside before moving into the BSC.
3.Place the cage into sanitized BSC and spray Clidox onto the outside surface of the cage and wipe with Clidox towel.
4.Take an autoclaved microisolator cage (has HEPA filter on top of the cage) into the BSC. If necessary, place a preheated heating pad into the cage to keep neonatal mice warm.
5.Open both cages and put the lids leaning against cage with inner side (clean side) out.
6.Transfer neonatal mice using long forceps one by one from holding cage to the transfer cage, and then close both cages. A preheated heating pad may be a necessity in cold weather.
7.Put the transferring cages into a secondary container with locking lid, close the secondary container, spray 70% ethanol on the outside of the secondary container.
8.Properly transport the secondary containers into designated handling rooms, and take out a cage from the secondary container.
Setting up apparatus
9.Prepare a BSC Class II for handling by clearing the BSC of any unnecessary objects and sterilizing the surface with Clidox and 70% EtOH as described above.
10.Take a large autoclaved microisolator cage for rat with HEPA filter on the top into BSC. Place a heating pad into the cage and a thermometer. Close the cage and adjust the temperature with the heating pad temperature settings. Find one that can keep the inner cage temperature at 29°C–31°C.
11.Place a water-resistant surface protector pad at the bottom of a cardboard or plastic box, and divide the box properly with cardboard. The size of the box and grid are based on the numbers of neonatal mice needed and their sizes for the experiments (Fig. 1). Usually 3 × 3 × 3 cm3 would be sufficient for two postnatal day 1 (P1) baby mice. The sizes can be adjustable based on the sizes of the pups. It is recommended to have a 1 cm gap between neonates and cotton balls. The height of the grid should be higher enough to prevent the escape of the mice from the grid. Place the boxes into the cage in BSC.
12.Milk powders (Enfamil ProSobee Soy Sensitive Baby Formula, Dairy-Free Lactose Free Plant Protein Milk Powder) are dissolved in autoclaved water at approximately 10% concentrations. Using a large petri dish, place cotton balls insider (Equate Beauty Jumbo Cotton Balls), and soak the cotton balls with milk. Based on the sizes of cotton balls, 5–10 ml milk per cotton ball is sufficient. A cotton ball can be split into several smaller pieces. Place 2–4 pieces of cotton balls soaked with soymilk into the corners of the grids described in previous step.
13.Place neonatal mice into the grids. It is recommended to put no more than two neonatal mice in one grid (Fig. 1).
14.Close the cage lid, change the cotton balls twice a day. The pad may be changed every day if necessary.
15.Manipulate and dispose the neonatal mice for proper experiments following approved protocols for desired times.
16.Spray the mouse cages with 10% bleach and/or 70% ethanol. Discard all the materials/objects into a biosafety waste container.
17.Remove the waste liquid container from the hood and add additional 10% bleach, wait for 10 min, and pour the waste liquid down the sink. Autoclave the plastic and other solid waste in a biohazard container and then dispose them as instructed by your institute’s biosafety officer.
18.Clean any equipment or surfaces that came in contact with the procedures with 10% bleach and then 70% ethanol.
19.Discard lab bench paper into biohazard bin and wipe down all surfaces and instruments that may have come in contact with the baby mice. Discard gloves.
DISCUSSION AND TROUBLESHOOTING
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