Figure 8

Induction of membrane translocation by serum depletion. The apoptosis-related membrane translocation assay was performed using a commercial kit (Accurate Chemical Scientific Corp.), which measured the uptake of a specific dye by cells as a result of apoptosis-induced membrane phosphatidylserine and phosphatidylcholine translocation. The assay was performed in 96-well microtiter plates. Cells were grown to confluence and followed by thoroughly washing with twice with PBS to remove serum, and then exposed to media with or without 10% serum for 1 1/2 hrs. At the end point, each well was washed twice with PBS and then exposed to the apoptosis dye reagent in DMEM with or without 10% FBS for 30 min. The cells were then photographed after the removal of dyes and then followed by PBS washing. (A) Control cells exposed to DMEM-10% FBS and 5 mM H₂O₂ for 1 1/2 hrs and followed by a 30 min-exposure to the same media containing the apoptosis marker dye. (B) Cells exposed to serum-free media for 1 1/2 hrs and then culture media containing both 10% FBS and the apoptosis dye marker for 30 min. (C) Cells exposed to serum-free media for 1 1/2 hrs and then serum-free media containing the apoptosis dye marker for 30 min.