Rhodamine 123 was used to stain and analyze by flow cytometry the mitochondria of rabbit articular chondrocytes. Stationary primary cultures and exponentially growing subcultures were compared to enzymatically released chondrocytes from cartilage. The increase in mitochondrial fluorescence, when chondrocytes are transferred from cartilage to culture environment, is suggestive of some change in chondrocyte adaptation andlor differentiation in these conditions. Key terms: Flow cytornetry, mitochondria, cell cycle, cell volumeIn most eukaryotic cells under normal aerobic conditions, the balance between respiratory and glycolytic metabolisms is related to growth and differentiated activities. Owing to their central role, mitochondria are sensitive indicators of this energy metabolism, and, moreover, their structure, activity, and biogenesis respond to physiological and pathological changes (6,16,17). However, why chondrocytes thrive, having poor but definite mitochondrial activities, in low oxygen tensions that other cell types would not tolerate and how this is related to the onset of their specific differentiation is not clearly understood (13,141. This prompted us to investigate possible changes in rnitochondrial metabolism as chondrocytes resume active cell multiplication when grown in vitro.Rhodamine 123, a positively charged fluorochrome at physiological pH, is able to stain mitochondria directly and provides low-background, high-fluorescence images of mitochondria. Use of this dye enables the selective staining of mitochondria in living cells and gives a good measure of mitochondria activity when quantified by flow cytometry (3,101.We have taken advantage of the specific mitochondria1 uptake of rhodamine 123 by living cells to re-examine the overall control of rnitochondrial activities in chondrocytes just released from cartilage as compared with cultured ones.
MATERIALS AND METHODSCell Culture Chondrocytes were enzymatically released from articular cartilage of 1-to-3-month-old "Fauve de Bourgogne" rabbits using a modification of Green's technique (7,ll).Isolated cells were then cultured in HAM F12 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (I.B.F., France), antibiotics (penicillin: 20 IU/ ml, streptomycin: 10 pg/ml, Special, France) and maintained at 37°C in an atmosphere of 75% N2, 20% 02, and 5% COz. When the monolayers reached confluence, cells were trypsinized and the first passage then obtained. Chondrocytes were seeded in 35-mm Petri dishes (7. lo4 cells) for microscopic examination and growth curves, and in 25-cm2 flasks (3.105 cells) for flow cytometric analysis.
Cell SizingElectronic aperture impedance was used as a signal for cell volume measurements.The system consists of a Coulter counter (model ZBI) and a pulse height analyzer (Coulter Channel AnalyserCoultronics). Cell volume was calculated after determination of the distribution maximal channel.Rhodamine 123 Uptake Rhodamine 123 (laser dye purity) was purchased from Eastman Organic Chemicals (Rochester, NY). The s...