Z vad fmk

We examined how long ovaries removed from a living body remain ready for transplantation using rat ovaries. As donor rats, slc: SD female rats, homebred and Crl: CD rats purchased from Jackson Laboratory Japan, aged over 8 weeks were used (n = 15). They were anesthetized with CO₂ inhalation and euthanized by cervical dislocation, then ovaries were removed. The removed left and right ovaries were each divided into four equal portions and stored in tissue preservation solution Lifor (Lifeblood Medical Inc., NJ, USA). During storage, they were kept at 4 °C in a tissue transport kit (CARD refrigerated transport kit, Kyudo Company, Japan), assuming marmoset ovary transport. The ovaries were implanted under the renal capsule of nude mice at 0, 1, 2, 3, and 4 days after the storage.
For ovarian protection experiments, Z-VAD-FMK (Caspase Inhibitor Z-VAD-FMK; G7231, Promega, USA) was added to Lifor (final concentration: 50 µM).

Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) cytotoxicity assay (TaKaRa, Shiga, Japan) and cell counting kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan), respectively, by following the manufacturer’s instructions. The cells were treated with 2% Triton X-100 as a positive control for the LDH cytotoxicity assay. The cells were then seeded at a density of 1 × 10⁴ cells/well in a 96-well cell culture plate in 10% FBS Ham’s F-12 K medium. After 24 h of incubation, the cells were treated with different concentrations of NYT in 1% FBS Ham’s F-12 K medium for 24 h, and then 5% CSE with NYT was added. After 24 h of incubation, the cell culture plates were centrifuged at 250 g for 10 min, and the supernatant was collected for performing the LDH assay. The LDH cytotoxicity assay kit was used to examine the level of LDH release, and the optical density of the samples was measured using a microplate reader Synergy H1 (BioTek, Tokyo, Japan) at 490 nm. CCK-8 was used to examine the cell viability, and the optical density was measured using a microplate reader set at 450 nm. To investigate the type of cell death that was induced by CSE exposure, the cells were treated with Z-VAD-FMK (Promega, Madison, WI, USA) or necrostatin-1 (Sellmeck Chemical, Houston, TX, USA) 2 h before the CSE exposure.