Detection of rubella-virus-induced apoptosis in Vero cell cultures with hematoxylin and eosin staining.
In order to facilitate the detection of apoptotic cells (Apo C) in Rubella virus (RV) infected cultures in settings of low resources, we compared hematoxylin and eosin staining (H&E) with the conventional TUNEL technique, and confirmed our findings with DNA electrophoresis and transmission electron microscopy. H&E allowed to distinguish Apo C from non-apoptotic cells. The proportion of Apo C in infected cultures was proportional to the multiplicity of infection (MOI). At a MOI of 10, the percent of Apo C at 3, 4 and 5 days post infection (pi) were 26, 45 and 47%, respectively, which were significantly reduced when the caspase inhibitor z-VAD-fmk was present in the supernatant. By the TUNEL assay, the percent of Apo C in RV-infected cultures were lower (0.8, 1.2 and 1.2% at 3, 4 and 5 days pi, respectively). Our results have shown that H&E staining is an easy, rapid, economic and reproducible method to detect Apo C in RV infected Vero cells cultures. It is possible that H&E makes evident early stages of apoptosis, when an apoptotic cell shows chromatin condensation, nuclear and cytoplasmic contraction (but is still attached to the monolayer), while TUNEL detects later stages of apoptosis because it needs an extensive DNA fragmentation, when apoptotic cells are about to or have already detached from the substratum.
Adamo
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MP
Laboratorio de Inmunología, Instituto de Virología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Agencia 4, Ciudad Universitaria, 5016 Córdoba, Argentina. marpilar@medinews.com
León Monzón
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M
Cuffini
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Pedranti
M
M
Zapata
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D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000502:physiology; D002448:Cell Adhesion; D002452:Cell Count; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000737:chemistry; D004396:Coloring Agents; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003588:Cytopathogenic Effect, Viral; D053938:DNA Fragmentation; D004801:Eosine Yellowish-(YS); D006416:Hematoxylin; D020287:In Situ Nick-End Labeling; D008854:Microscopy, Electron; D015203:Reproducibility of Results; D012412:Rubella virus / Q000502:physiology*; D013194:Staining and Labeling / Q000191:economics / Q000379:methods*; D014709:Vero Cells / Q000737:chemistry / Q000648:ultrastructure / Q000821:virology*
D000590:Amino Acid Chloromethyl Ketones; D002843:Chromatin; D004396:Coloring Agents; D015853:Cysteine Proteinase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases; D004801:Eosine Yellowish-(YS); D006416:Hematoxylin