We further analyzed neurons that were rescued from apoptosis by these caspase inhibitors, and discovered that these neurons are vulnerable to subsequent stimuli that produce Ca2q influx. The following reagents used in the present experiments were ordered from the individual companies explained below: Fetal bovine serum FBS., Moregate Australia., Eagles minimum crucial medium MEM., Nissui Tokyo, Japan., other cell culture media and reagents, Gibco Grand Island, NY., human recombinant brain derived neurotrophic factor BDNF., Pepro Tech Rocky Hill, NJ., acetyl Asp Glu Val Asp 4 methylcoumaryl 7 amide Ac DEVD MCA., Ac Everolimus mTOR inhibitor DEVD CHO, acetyl Tyr Val Ala Asp 4 methylcoumaryl 7 amide Ac YVAD MCA., and Ac YVAD CHO, Peptide Institute Osaka, Japan., Z Asp 2,6 dichlorobenzoyloxy methylketone Z Asp CH DCB., Funakoshi Tokyo, Japan., Boc Asp fluorometh 2 ylketone Boc Asp FMK., Z Val Ala Asp fluoromethylketone Z VAD FMK., and Z Asp Glu Val Asp fluoromethylketone Z DEVD FMK., Enzyme Systems Products Dublin, CA., human recombinant CPP32 with C final His tag., 6 Upstate Biotechnology Lake Placid, NY., assay products to ascertain cellular reduction activity of MTT, 4 w3 4 iodophenyl. 2H 5 tetrazoliox 1,3 benzene disulfonate WST 1., and sodium 3X w1 phenylamino carbonyl. 3,4 tetrazoliumxbis 4 methoxy 6 nitro. benzenesulfonic acid hydrate OR., assay kit for determination of LDH exercise, Boehringer Mannheim Mannheim, Germany., propidium iodide PI., Molecular Probes Eugene, XTT., calcein acetoxymethyl ester calcein AM., Wako Osaka, Japan., and Lymph node the others, Sigma St. Louis, MO.. Primary cultures of cerebellar granule neurons were obtained from dissociated cerebella of 7 to 8 day old Sprague? Dawley rats, as described previously w15x. In brief, after being eliminated, cerebella were dissected, and trypsinized at 378C. The cells were seeded onto 48 effectively culture plates precoated with poly D lysine, in a density of 3?4 105 cellsrcm2 in basal changed Eagles medium containing 25 mM KCl, 10% FBS, 2 mM glutamine and 50 mgrml gentamicin. Cytosine arabinofuranoside 10 mM. was added 18?24 h after plating to inhibit the development of non neuronal cells. Five to 6 days after plating, the culture medium was removed, washed once, and replaced with serum free MEM containing 5. 6 mM KCl low KCl. with or without drugs. Being a get a handle on, some wells in each dish were washed and changed with Dinaciclib CDK Inhibitors MEM containing 25 mM KCl high KCl., and some wells were left alone without medium change intact cells.. Double staining of both viable and dead neurons was used. Cells were incubated at 378C for 30 min with MEM containing 1 mM calcein AM, which is cleaved by esterases present in living cells glowing yellow green fluorescence, and 10 mgrml PI, which is adopted in dead cells and becomes orange red fluorescent by intercalation in to DNA.