This study was carried out in accordance with the recommendations of PHS Policy on Humane Care and Use of Laboratory Animals, the Guide for the Care and Use of Laboratory Animals, and the policies and procedures of the University of Central Arkansas. The protocol was approved by the UCA Animal Care and Use committee.
Mouse DRG were collected from embryonic day 12.5 (E12.5) CD1 mice embryos. The DRGs from 5 embryos (~200 total DRGs) of CD1 mice were dissociated and resuspended in 50 μl of Neurobasal media (Invitrogen) containing 2% B27 (Invitrogen) and 50 ng/ml NGF (Harlan Bioproducts) per dissected embryo. Suspended DRG neurons were placed as a drop (2 μl) near one end of each well in Lab TekII 4-well chambered cover glass (Nalge Nunc International), which had been previously coated with poly-D-lysine (0.1 mg/ml) and laminin (2–5 ug/ml). The chambered cover glass was then incubated at 37°C at 5% CO2 for 15 min before 500 μl of Neurobasal media containing 2% B27, 50 ng/ml NGF, 1 μm 5-fluro-2′-deoxyuridine (Sigma), and 1μm uridine (Sigma) was added to each well.
Vincristine (0.4 μM) was added to 14 days in vitro (DIV) cultures to induce axon degeneration.
Mouse DRG were collected from embryonic day 12.5 (E12.5) CD1 mice embryos. The DRGs from 5 embryos (~200 total DRGs) of CD1 mice were dissociated and resuspended in 50 μl of Neurobasal media (Invitrogen) containing 2% B27 (Invitrogen) and 50 ng/ml NGF (Harlan Bioproducts) per dissected embryo. Suspended DRG neurons were placed as a drop (2 μl) near one end of each well in Lab TekII 4-well chambered cover glass (Nalge Nunc International), which had been previously coated with poly-D-lysine (0.1 mg/ml) and laminin (2–5 ug/ml). The chambered cover glass was then incubated at 37°C at 5% CO2 for 15 min before 500 μl of Neurobasal media containing 2% B27, 50 ng/ml NGF, 1 μm 5-fluro-2′-deoxyuridine (Sigma), and 1μm uridine (Sigma) was added to each well.
Vincristine (0.4 μM) was added to 14 days in vitro (DIV) cultures to induce axon degeneration.