Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants

Abstract

Summary:Apoptosis is the common cell death pathway which is initiated by a variety of different stimuli. The recognition of early apoptotic events would markedly improve reliability and convenience of apoptosis assays. In the present study the vital stain Syto R 16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-AAD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V. The method was established using solid tumour cell lines treated with TNF. Subsequently we applied it to determine apoptotic populations in CD34 + peripheral blood progenitor cells obtained from growth factor and/or chemotherapy mobilised patients and frozen/thawed according to standard stem cell transplantation protocols.

Towards an understanding of apoptosis detection by SYTO dyes

“…For multiparametric (two-color and tri-color stainings) logarithmic fluorescence signals were collected using following filters: SYTO dyes (FL1 530/30 BP), TMRM (FL2 585/42 BP), PI (FL2 585/42 BP), 7-AAD (FL3 650 LP). As described by others (14) due to the strong spectral overlap of SYTO16 (in FL2 and FL3) special attention has been made to compensate both channels. For evaluation of fractional DNA content propidium iodide fluorescence was collected using linear amplification scale and 585/42 BP filter.…”

“…It has been previously tested by others that loss of SYTO16 fluorescence can be considered as a truly apoptotic feature (12)(13)(14)23,24), but to the best of our knowledge there is very limited data available to show SYTO behavior in cells exposed to oncotic stimuli. We observed that in the presence of relatively harsh oncotic stimuli (4 h-incubation with 1% NaN 3 , or 5 min at 56°C) the majority of cells was SYTO low /PI 1 , representative of cells with a compromised plasma membrane, whereas the number of SYTO dim /PI 2 events did not significantly increase above background levels ( Fig.…”

“…Following on from Fray's publication back in 1995, cell permeable SYTO probes have been gaining momentum as reliable discriminators of live, apoptotic, and dead cells, offering cost-effective and easy to perform assays for tracking apoptosis in cultured and primary cells, and proving amenable for the development of multiparameter flow cytometry assays (11)(12)(13)(14)(15)(16). Dyes from SYTO families (blue-, green-, orange-, and red-fluorescent) are generally permeable (although at varying levels) to all cell membranes, including bacterial.…”

“…It should be noted, however, that SYTO probes are not exclusive DNA stains, and thus several of them have been successfully applied to visualize translocation of RNA granules in neurons and allow differential discrimination of nuclear/mitochondrial DNA from cytoplasmic RNA using two-photon lifetime imaging (17,22). Importantly recent reports suggest the reliability of some SYTO dyes as effective substrates in quantitative P-glycoprotein (P-gp) function assessment (14,19,23).…”

“…Although mounting evidence show higher sensitivity of SYTO probes as compared with Annexin V based assays (12,14,24), the exact mechanisms underlying SYTO differential staining of apoptotic and viable cells still remain ambiguous. The most widely embraced idea is the self-quenching of SYTO dye molecules following changes in interprobe proximity or the decrease in SYTO binding sites as the chromatin condensation and RNA degradation advance in the process of apoptosis (11,13,17).…”