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Quantification of cellular viability by automated microscopy and flow cytometry.

Sauvat A, Wang Y, Segura F, Spaggiari S, Müller K, Zhou H, Galluzzi L, Kepp O, Kroemer G - Oncotarget (2015)

Bottom Line: However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death.Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels).This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.

View Article: PubMed Central - PubMed

Affiliation: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.

ABSTRACT
Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.

No MeSH data available.


Related in: MedlinePlus

Flow cytometry-based assessment of cellular viability to standard inducers of apoptosis(A–C) Human non-small cell lung carcinoma A549 cells were cultured in control conditions or treated with 500 μM oxaliplatin or 4 μM staurosporine for 24 hrs, then co-stained with DAPI, DiOC6(3) and YO-PRO®-3 and analyzed by flow cytometry. Representative dot plots are reported. In panels A, B and C, numbers indicate the percentage of events within each gate. See also Supplementary Figure 2.
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Figure 2: Flow cytometry-based assessment of cellular viability to standard inducers of apoptosis(A–C) Human non-small cell lung carcinoma A549 cells were cultured in control conditions or treated with 500 μM oxaliplatin or 4 μM staurosporine for 24 hrs, then co-stained with DAPI, DiOC6(3) and YO-PRO®-3 and analyzed by flow cytometry. Representative dot plots are reported. In panels A, B and C, numbers indicate the percentage of events within each gate. See also Supplementary Figure 2.

Mentions: Next, we implemented the triple staining protocol described above on a cytofluorometric platform amenable to automation. To this aim, human NSCLC A549 cells maintained in control conditions or exposed to lethal stimuli for 24 hrs were collected and then co-stained with DAPI, DiOC6(3) and YO-PRO®-3. The vast majority of untreated A549 cells exhibited a DAPI−DiOC6(3)high YO-PRO®-3− phenotype, indicating intact plasma membranes, normal mitochondrial functions and no caspase activation (Figure 2A and Supplementary Figure 3). In contrast, exposing A549 cells to 500 μM oxaliplatin or 4 μM staurosporine for 24 hrs caused the accumulation of DAPI+ cells, as well as of cells exhibiting a DAPI−DiOC6(3)lowYO-PRO®-3+, DAPI−DiOC6(3)highYO-PRO®-3+ or DAPI−DiOC6(3)low YO-PRO®-3− staining pattern (Figure 2B, 2C and Supplementary Figure 3). Hence, both oxaliplatin and staurosporine cause a drop in the absolute amount of DAPI−DiOC6(3)high YO-PRO®-3− (viable) A549 cells in a dose-dependent fashion (Figure 3A). The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (Z-VAD-fmk) was unable to prevent the loss of viability among cells cultured in the presence of oxaliplatin or staurosporine. However, caspase inhibition with Z-VAD-fmk substantially reduced the surge in DAPI−DiOC6(3)lowYO-PRO®-3+ and DAPI−DiOC6(3)highYO-PRO®-3+ cells, without eliminating the DAPI−DiOC6(3)lowYO-PRO®-3− subset. This was particularly evident when all events in control, oxaliplatin or staurosporine-treated conditions were analyzed upon aggregation, as if they derived from a single sample, either in the absence (Figure 3B, left panels) or in the presence of Z-VAD-fmk (Figure 3B, right panels). These results confirm the stringent requirement for caspase activation for the opening of YO-PRO®-3-permeable PANX1 channels [19, 20], and validate the feasibility and sensitivity of our viability test.


Quantification of cellular viability by automated microscopy and flow cytometry.

Sauvat A, Wang Y, Segura F, Spaggiari S, Müller K, Zhou H, Galluzzi L, Kepp O, Kroemer G - Oncotarget (2015)

Flow cytometry-based assessment of cellular viability to standard inducers of apoptosis(A–C) Human non-small cell lung carcinoma A549 cells were cultured in control conditions or treated with 500 μM oxaliplatin or 4 μM staurosporine for 24 hrs, then co-stained with DAPI, DiOC6(3) and YO-PRO®-3 and analyzed by flow cytometry. Representative dot plots are reported. In panels A, B and C, numbers indicate the percentage of events within each gate. See also Supplementary Figure 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496231&req=5

Figure 2: Flow cytometry-based assessment of cellular viability to standard inducers of apoptosis(A–C) Human non-small cell lung carcinoma A549 cells were cultured in control conditions or treated with 500 μM oxaliplatin or 4 μM staurosporine for 24 hrs, then co-stained with DAPI, DiOC6(3) and YO-PRO®-3 and analyzed by flow cytometry. Representative dot plots are reported. In panels A, B and C, numbers indicate the percentage of events within each gate. See also Supplementary Figure 2.
Mentions: Next, we implemented the triple staining protocol described above on a cytofluorometric platform amenable to automation. To this aim, human NSCLC A549 cells maintained in control conditions or exposed to lethal stimuli for 24 hrs were collected and then co-stained with DAPI, DiOC6(3) and YO-PRO®-3. The vast majority of untreated A549 cells exhibited a DAPI−DiOC6(3)high YO-PRO®-3− phenotype, indicating intact plasma membranes, normal mitochondrial functions and no caspase activation (Figure 2A and Supplementary Figure 3). In contrast, exposing A549 cells to 500 μM oxaliplatin or 4 μM staurosporine for 24 hrs caused the accumulation of DAPI+ cells, as well as of cells exhibiting a DAPI−DiOC6(3)lowYO-PRO®-3+, DAPI−DiOC6(3)highYO-PRO®-3+ or DAPI−DiOC6(3)low YO-PRO®-3− staining pattern (Figure 2B, 2C and Supplementary Figure 3). Hence, both oxaliplatin and staurosporine cause a drop in the absolute amount of DAPI−DiOC6(3)high YO-PRO®-3− (viable) A549 cells in a dose-dependent fashion (Figure 3A). The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (Z-VAD-fmk) was unable to prevent the loss of viability among cells cultured in the presence of oxaliplatin or staurosporine. However, caspase inhibition with Z-VAD-fmk substantially reduced the surge in DAPI−DiOC6(3)lowYO-PRO®-3+ and DAPI−DiOC6(3)highYO-PRO®-3+ cells, without eliminating the DAPI−DiOC6(3)lowYO-PRO®-3− subset. This was particularly evident when all events in control, oxaliplatin or staurosporine-treated conditions were analyzed upon aggregation, as if they derived from a single sample, either in the absence (Figure 3B, left panels) or in the presence of Z-VAD-fmk (Figure 3B, right panels). These results confirm the stringent requirement for caspase activation for the opening of YO-PRO®-3-permeable PANX1 channels [19, 20], and validate the feasibility and sensitivity of our viability test.

Bottom Line: However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death.Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels).This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.

View Article: PubMed Central - PubMed

Affiliation: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.

ABSTRACT
Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.

No MeSH data available.


Related in: MedlinePlus