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1.
Methods Mol Biol ; 1943: 301-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838624

RESUMO

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell-death related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of caspase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.


Assuntos
Poliaminas/toxicidade , Testes de Toxicidade/métodos , Transfecção/métodos , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Corantes Fluorescentes/química , Fluorometria/métodos , Humanos , Células Jurkat , Ácidos Nucleicos/genética , Polieletrólitos
2.
Methods Mol Biol ; 948: 23-33, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23070761

RESUMO

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.


Assuntos
Caspase 3/metabolismo , Caspase 7/metabolismo , Citotoxinas/toxicidade , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Poliaminas/toxicidade , Ensaios Enzimáticos/normas , Fluorometria/normas , Humanos , Indicadores e Reagentes/química , Células Jurkat , Polieletrólitos , Padrões de Referência
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