BackgroundTo study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin‐V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death.MethodsApoptosis was induced in Jurkat cells by γ‐irradiation, incubation with camptothecin (CPT), or cytosine β‐D‐arabinofuranoside (Ara‐C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze‐thaw procedure. Various AnV/PI− CD34+ fractions were cultured in a single‐cell single‐well (SCSW) assay.ResultsJurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI− cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80–90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI− fractions (overall range 23–62%). Within total AnV+ and AnV+/PI− populations, only a minority of CD34+ cells showed ISEL positivity (range 4–8% and 0.8–6%, respectively). Different fractions of AnV+/PI− CD34+ cells did have clonogenic capacity.ConclusionsPCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo‐nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV‐fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI− CD34+ cells in the SCSW assay. Cytometry 47:24–31, 2002. © 2001 Wiley‐Liss, Inc.

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Discussion about the treatment of hallux valgus is largely confined to the merits of various operations on the big toe itself, and the problem of associated metatarsalgia is given rather less detailed consideration. THE JOURNAL OF BONE AND JOiNT SURGERY

BackgroundNormal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP‐binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux.MethodsWe used epifluorescence microscopy (EFM) in combination with single‐cell image analysis to study ABC‐transporter–mediated efflux in highly purified, viable, CD34+CD38‐ cells sorted on an adhesive biolayer. P‐glycoprotein and multidrug‐resistant protein (MRP)‐mediated efflux were quantitated using fluorescent substrates (rhodamine‐123 and calcein acetoxymethyl ester [calcein‐AM]) and specific inhibitors (verapamil and probenecid, respectively).ResultsThe feasibility, sensitivity, and reproducibility of rhodamine‐123 efflux quantitation using single‐cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P‐glycoprotein–mediated transport was higher in CD34+CD38‐ cells than in more differentiated progenitors (mean efflux index = 2.24 ± 0.35 and 1.14 ± 0.11, respectively; P = 0.01). P‐glycoprotein–mediated transport was the main determinant of the rhodamine “dull” phenotype of these cells. In addition, significant MRP‐mediated efflux was demonstrated in CD34+CD38‐ and CD38+ cells (mean efflux index = 1.42 ± 0.19 and 1.28 ± 0.18, respectively).ConclusionThe described method is a valuable tool for assessing ABC‐transporter–mediated efflux in highly purified single cells. Both P‐glycoprotein and MRP‐mediated efflux are present in human CD34+CD38‐ hematopoietic stem cells. Cytometry 49:135–142, 2002. © 2002 Wiley‐Liss, Inc.

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