The effect of GM-CSF rescue from apoptosis on ceramide levels in TF-1 cells. TF-1 cells were treated with γ radiation (5 Gy) in either the presence or absence of GM-CSF (100 U/mL) in complete RPMI supplemented with 20% FBS, and harvested at 2, 4, 8, 10, and 60 minutes postirradiation. Extraction and measurement of ceramide was performed as described37 by assay of ³²P incorporated upon phosphorylation of ceramide to ceramide 1-P by DAG kinase. Ceramide 1-P was resolved by TLC using CHCl₃:CH₃OH:acetic acid (65:15:5, vol/vol) as solvent, detected by autoradiography and the incorporated³²P was quantified by phosphoimaging (Fugi BAS1000, Fugi Medical Systems). The level of ceramide was determined by comparison to a concomitantly run standard composed of known amounts of ceramide. Ratios were derived by comparison to ceramide levels of unirradiated TF-1 cells grown in either the presence or absence of GM-CSF. The values were derived from duplicate experiments and error bars represent standard deviations into which mean values were incorporated.