Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip.
BACKGROUND
This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required.


METHODS
Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer.


RESULTS
Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used.


CONCLUSIONS
Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.
Wolbers
F
F
Department of Clinical Chemistry, Medisch Spectrum Twente, Hospital Group, PO Box 50000, 7500 KA Enschede, The Netherlands.
Andersson
H
H
van den Berg
A
A
Vermes
I
I
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D002166:Camptothecin / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005240:Feasibility Studies; D005434:Flow Cytometry; D005453:Fluorescence; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014965:X-Rays
D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D002166:Camptothecin