Journal:

Article Title: Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine

doi:

Figure Lengend Snippet: Translocation-dependent decarboxylation of C6-NBD-PS in isolated mitochondria from CHO-K1 and R-41 mutant cells. Intact (A) or disrupted (B) mitochondria from either CHO-K1 or R-41 mutant cells were incubated with 10 μM C6-NBD-PS in 1 ml of buffer A (250 mM mannitol/5 mM Hepes, pH 7.4/0.5 mM EGTA) at 39.5°C for various periods. The reaction was terminated by adding 3.75 ml of chloroform/methanol, 1:2 (vol/vol), and then the lipids were extracted and separated by TLC. C6-NBD-PE spots were scraped from TLC plates, and then the lipids were extracted as described by Bligh and Dyer (26). The relative fluorescence of the lipid extracts was measured with a fluorescence spectrophotometer F-2000 (Hitachi, Tokyo). Mitochondria were disrupted by exposure five times to ultrasound on ice for 30 sec at setting 1 in a Branson ultrasonic disrupter. ○, CHO-K1; ●, R-41. R.F.U., relative fluorescent unit(s).

Article Snippet: The relative fluorescence of the lipid extracts was measured with a fluorescence spectrophotometer F-2000 (Hitachi, Tokyo).

Techniques: Translocation Assay, Isolation, Mutagenesis, Incubation, Fluorescence, Spectrophotometry