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Article History

Received: 8 August 2024

Accepted: 11 December 2024

First Online: 28 December 2024

Declarations

:

: The authors declare no competing interests.

: All experimental protocols were approved by the Department of Mechanical Engineering, Qilu University of Technology (Shandong Academy of Sciences) committee.All manipulations of cell passaging culture have been performed in strict accordance with the ATCC Animal Cell Culture Guide (). All experiments are strictly according to ARRIVE guidelines (). Mice were euthanized using CO₂ anesthesia combined with neck dissection, and we hereby declare that all of our handling methods were performed in strict accordance with the ARRIVE guidelines (). KM mice from 1 to 7 days old were selected for the experiment. The mice were kept in a comfortable environment with a temperature of 26℃ and a relative humidity of 55%. The mice were kept in a light and dark cycle for 12 hours, and were given adequate food and water (protein 25%, fat 8%, carbohydrates 55%, vitamins and minerals 12%). For KM mice from 1 to 7 days old, in order to reduce the pain of the animals, we took appropriate measures, and selected CO₂ euthanasia combined with neck amputation. We have invited professionally trained personnel to perform the required operations. We made sure that the CO₂ euthanasia box was clean and well sealed, that the mice were placed in the euthanasia box without being pre-infused with CO₂ gas, and that the density of the animals in the box was moderate and there was no overcrowding. Connect the CO₂ gas pipe, open the valve of the gas cylinder, and inject CO₂ gas at a rate of 10% to 30% of the volume of the euthanasia box replaced per minute. To observe the reaction of mice, after they faint and lose the ability to exercise, increase the flow of gas,but the maximum flow can not exceed 0.5KPa. After confirming that the mice were not moving, not breathing, and their pupils were dilated, the CO₂ gas was turned off and observed for another two minutes to ensure that the mice lost consciousness. Remove the mice from the euthanasia box and place them on a hard surface. Use your thumb and index finger to quickly press on the neck of the mouse while pulling hard on the tail of the mouse to dislocate its cervical vertebra. Confirm that the mouse is dead and that the procedure is painless. The skin was then rinsed with alcohol and an appropriate size of skin was clipped for further analysis. All operations are in accordance with ARRIVE guidelines. This research report in strict accordance with the ARRIVE guidelines, to ensure that involved in animal research transparent and repeatable.