Flow cytometry of apoptotic cell death

Vermes, I.; Haanen, C.; Reutelingsperger, Chris

doi:10.1016/s0022-1759(00)00233-7

Cited by 684 publications

(508 citation statements)

References 97 publications

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“…The samples can on the one hand be analyzed using a plate reader, but if information is requested on the variation between different cell types or status (e.g. apoptotic or not), flow cytometry is a more commonly used technique [86][87][88][89] . On the other hand microscopic analysis can also be performed.…”

Section: A Routine In Vitro Methodsmentioning

confidence: 99%

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

et al. 2013

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Section: A Routine In Vitro Methodsmentioning

confidence: 99%

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

et al. 2013

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“…Early apoptosis is a preferred indicator of AICD because cells that have progressed to a PI ϩ phase are readily engulfed by macrophages, and thus not accessed in lymphocyte gates. Furthermore, late-stage apoptotic cells cannot be distinguished from those that are necrotic as a consequence of cellular injury rather than programmed cell death (57). Early apoptotic cells are less likely to be phagocytosed than the late apoptotic cells.…”

Section: Liver-derived Virus-specific Cd8 ϩ T Cells Are Viablementioning

confidence: 99%

Virus-Specific CD8+ T Cells in the Liver: Armed and Ready to Kill

Keating

Yue

Rutigliano

et al. 2007

The Journal of Immunology

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Influenza A virus infection of C57BL/6 mice is a well-characterized model for studying CD8+ T cell-mediated immunity. Analysis of primary and secondary responses showed that the liver is highly enriched for CD8+ T cells specific for the immunodominant H2DbNP366–374 (DbNP366) epitope. Functional analysis established that these liver-derived virus-specific CD8+ T cells are fully competent cytotoxic effectors and IFN-γ secretors. In addition, flow cytometric analysis of early apoptotic cells showed that these influenza-specific CD8+ T cells from liver are as viable as those in the spleen, bronchoalveolar lavage, mediastinal lymph nodes, or lung. Moreover, cytokine profiles of the influenza-specific CD8+ T cells recovered from different sites were consistent with the bronchoalveolar lavage, rather than liver population, being the most susceptible to activation-induced cell death. Importantly, adoptively transferred influenza virus-specific CD8+ T cells from the liver survived and were readily recalled after virus challenge. Together, these results show clearly that the liver is not a “graveyard” for influenza virus-specific CD8+ T cells.

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“…Many different markers to detect distinct time points of apoptotic pathways are available for detection of apoptosis (14,15). However, all of the methodologies offered on the market include at least one step of cell washing which, according to our experience, is not in line with quantitative cell analysis.…”

Section: Discussionmentioning

confidence: 99%

Novel single‐platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no‐wash procedure

et al. 2010

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FCM is a generally accepted tool to analyze apoptosis. Unfortunately, the cell preparation of all commercial kits available includes cell washing known to cause cell loss which is most likely to affect apoptotic cells in particular. To address this, we developed a seven-color single-platform no-wash analysis technique and compared the results with those from an analogous procedure including cell washing. A five-color mAb cocktail was employed to address target cells by surface labeling, Yo-PRO-1 1 and DAPI were used to discriminate apoptotic and necrotic from viable cells. Cells were quantified on the basis of internal-standard fluorescent beads. Jurkat cells ACC 282 treated with camptothecin were employed to establish the staining procedure, which was then applied to blood cells collected by extracorporeal apheresis and treated with UV irradiation. Data evaluation showed that although each method by itself was highly reproducible (R 2 5 0.973), the numbers of apoptotic cells detected with the no-wash procedure were significantly higher than those obtained after cell washing (P 5 6.6 E 25 , Wilcoxon Test). In addition, the observed differences increased with higher cell numbers (Bland and Altmann). We conclude that the described test is a feasible and reliable tool for apoptosis measurement and it provides results that are definitely closer to the truth than those obtained from kits that require cell washing. ' 2010 International Society for Advancement of CytometryKey terms multicolor flow cytometry; apoptosis; Trucount TM ; single platform; Yo-PRO-1 1 APOPTOSIS plays a major role in various physiological and pathological processes.Cell demise has been investigated in different fields, also including extracorporeal photopheresis (ECP), a therapeutic intervention used in several auto immune disorders, diseases with pathogenic T cell involvement, and chronic graft-versus-host disease (GvHD) (1). Collected cells are reinfused after in vitro exposure to 8-methoxypsoralen and ultraviolet A (UVA) light. One known effect of ECP on PBMC is the induction of apoptosis (2,3), which can be analyzed by FCM, a generally accepted and reliable tool for detection of apoptosis on single-cell level (4,5). Several assays for FCM analysis of different stages of programmed cell death are commercially available but all of them include at least one centrifugation step. Hypothesizing that, after apoptosis-inducing treatment, part of the PBMC will be disrupted by centrifugation and thus will not be present for FCM analysis, our intention was to establish a single-platform, no-wash, and multicolor FCM methodology to quantify apoptotic T cells in ECP products.To discriminate viable from early and late apoptotic/necrotic cells, we tested several markers. We favored the YO-PRO-1 1 /DAPI combination which allows discrimination of apoptotic from necrotic cells. YO-PRO-1, a DNA-intercalating dye excited at 488 nm, is a reliable marker for FCM analysis of apoptosis (6,7). DAPI, a nuclear dye excited at 405 nm, which is commonly used in ...

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Flow cytometry of apoptotic cell death

Cited by 684 publications

References 97 publications

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

Virus-Specific CD8+ T Cells in the Liver: Armed and Ready to Kill

Novel single‐platform multiparameter FCM analysis of apoptosis: Significant differences between wash and no‐wash procedure

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