Silicon grease

COCs were obtained from the mated females used for ejaculated sperm collection. Both oviducts were removed and placed in 500 μl medium in a petri dish right before dissecting the uterus of the mated female for sperm recovery. The large cumulus mass was recovered from each oviduct by tearing open the wall of oviduct with a 27g needle. The cumulus mass was gently separated into COCs containing 2–4 oocytes each. A COC was placed on the 14 mm diameter recessed glass insert of a 35 mm petri dish (MatTek Corporation, Ashland, MA; Part Number: P35G-0-14-C) and covered with a 3 mm x 3 mm coverslip supported by four dabs of silicon grease (Corning Inc., Corning, NY) (Fig 2A, arrow). As a negative control, four dabs of silicon grease supporting a coverslip were placed in the same recessed insert (Fig 2A, arrowhead). A total of 50 μl medium was added into the insert and then covered with mineral oil. The observation dish was incubated at 37°C under 5% CO₂ in humidified air for at least 1.5 h before addition of sperm.

Single cells with space of 2 microscopic view fields were selected for clonal culturing. To separate single cells, clonal cylinders (Sigma-Aldrich, Munich, Germany) were placed. The cylinders were coated with silicon grease (Corning, Wiesbaden, Germany) on the lower site to eliminate medium exchange between clonal cultures and the rest of the plate. The success of isolating a single cell was confirmed microscopically. Non-adherent cells were removed by exchange of medium. Clonal growing periosteal cells were cultured under standard cell culture conditions and the medium was replaced every 2–3 days in the cylinders. When reaching about 90% confluence, clonal cultures were sub-cultured by trypsin-EDTA (Biochrom) treatment (0.5%) and subsequently replated in a well of a 6-well plate and further cultivated.