Cytofluorometric Quantitation Of Apoptosis-driven Inner Mitochondrial Membrane Permeabilization With Calcein-AM

BACKGROUND AND OBJECTIVE:Many research have demonstrated that mitochondria were important in cell apoptosis regulation, and inner mitochondrial membrane permeabilization constituted an early event in the apoptotic cascade. The mitochondrial matrix can be specifically labeled by loading cells with calcein fluorescence with cobalt(Co2+).Positive staining of mitochondrion thus requires that the inner mitochondrial membrane functions as a barrier separating calcein(within the matrix) from Co2+(outside of the matrix).The current study was designed to estabilish a new method for early detection of apoptosis by Cytometry.METHODS:The Molt-4 cell line loaded with calcein-AM and cobalt(Co2+) were treated with Camptothecin(CPT) or ultraviolet(UV),then analyzed the diversification of calcein fluorescence by cytometry in certain time after apoptosis inducing. RESULTS:(1) The overall calcein fluorescence decreased after apoptosis inducing, whether induced with Camptothecin(CPT) or ultraviolet(UV); (2)The calcein fluorescence decreased in time-dependent pattern after certain method of apoptosis inducing;(3) The calcein fluorescence decreased in dose-dependent pattern after certain method of apoptosis inducing; CONCLUSION:Calcein-AM is a kind of sensitive vital maker of apoptosis, and it can be used to detect early apoptosis by cytometry. OBJECTIVE:The current study was designed to compare the sensitivity of Calcein-AM and Annexin V-FITC for early detection of apoptosis in living cells by Flow Cytometry and to estabilish a new method for early detection of apoptosis.METHOD:The Molt-4 cell line treated with camptothecin(CPT) or ultraviolet(UV) were labeled with Calcein-AM and FITC-conjugated Annexin V respectively, then analyzed the apoptosis rate by flow cytometry and compared the performance of Calcein-AM and Annexin V for early detection of apoptosis.RESULTS:Our results show that both probes allowed the detection of apoptotic cells. However, Calcein-AM was more sensitive than Annexin V. We could detect the apoptosis induced by CPT two hours after inducing with Calcein-AM but three hours after inducing with Annexin V. And, the apoptosis induced by UV could be detected one hours after inducing with Calcein-AM but two hours after inducing with Annexin V.CONCLUSION:Annexin V is less sensitive than Calcein-AM for early apoptosis detection, and the new method detecting early apoptosis with Calcein-Am is stable, reliable, sensitive and low-cost.