CYTOTOXIC Drug Treatments

Notes: + all treatments have been optimized for use in mouse/rat primary neurons at 5-7 days in-v itro (DIV.)

Glutamate/Glycine induced excitoxicity (TOCRIS #0218,0219)

  • Make 10mM stock for Glu, 100mM stock for Gly. Gly is always treated at 1/10th  amount of Glu.
  • Usually used concentrations are 100uM or 1mM, however
  • Acute excitotoxicity in semi-mature: 1mM Glu only. 1mM Glu will kill ~50% of DIV6 lipo’ed mouse corticals in 16~20h. Later DIVs likely faster.

Hydrogen Peroxide (Mallin ckrodt, #5240 (30% Solution = 8.82M))

  • Make 10mM or 100mM stock (100mM = 23ul in 2ml dH2O).
  • 25uM can generate p25
  • 100uM can robustly induce cell death, but dropoff seen from 1h.

Etoposide DNA synthesis inhibitor/ topoisomerase I inhibitor / induces DSBs (Sigma #E1383)

  • Make 1mM stock in DMSO (5.9mg in 10mL)
  • Treat at 1uM, 12~24h
  • 5uM, 12h will give you excellent H2AX, moderate cell death (~40%)
  • 2.5uM for 24h will cause ~80% cell death?
  • Try 10uM, 20uM for 12h; some papers go as high as 100uM

Camptothecin REVERSIBLE topoisomerase inhibitor / induces DSBs (Sigma, C9911)

  • Dissolve in DMSO
  • Furuta, Hayward, Meng, et al: 1uM for 3 hours results in H2AX; will subsequenty decrease at 3h and 6h post removal. Thus, susceptibility to and subquent removal of H2AX can be measured by Western, staining.
  • Treat 10uM, 12 hours to see ~50% drop in viability