Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy

Our plain-English summary of Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy, by W.P. Davis, Y.M. Janssen, B.T. Mossman, et al (Histochemistry and Cell Biology, 1997).

The study

• Authors: W.P. Davis, Y.M. Janssen, B.T. Mossman, D.J. Taatjes
• Journal: Histochemistry and Cell Biology
• Published: 1997;108(4-5):307-11
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

We describe a multifluorescence labeling technique for simultaneous detection of mRNA, nuclear DNA, and apoptosis in cultured cells. Digoxigenin-labeled cRNA probes were used to study proto-oncogene expression in rat pleural mesothelial cells undergoing apoptosis following exposure to crocidolite asbestos or hydrogen peroxide (H2O2). Hybridized cRNA probe was detected by immunolocalization with an anti-digoxigenin monoclonal primary and fluorophore-conjugated anti-mouse secondary antibody. Cells undergoing apoptosis were simultaneously identified by the TdT-mediated biotin-dUTP nick-end labeling (TUNEL) method and a streptavidin-conjugated far-red fluorophore, and nuclear DNA was stained with oxazole yellow dimer (YOYO-1). With confocal scanning laser microscopy, we demonstrated increased c-jun mRNA expression within the cytoplasm of both TUNEL-positive and non-apoptotic cells following exposure to either crocidolite asbestos or H2O2. Thus, this technique represents a useful in vivo approach for evaluating apoptosis-associated gene expression with confocal scanning laser microscopy.

In plain English

This laboratory methods paper described a way to stain cultured cells with three fluorescent labels at once and view them with a confocal laser microscope. The labels showed messenger RNA (mRNA) for the c-jun gene, the DNA in each cell's nucleus, and cells undergoing apoptosis (a form of programmed cell death), detected by a method called TUNEL. The authors tested the method on pleural mesothelial cells taken from adult Fisher 344 rats and grown in culture. The cells were exposed to crocidolite asbestos (5.0 µg/cm2) for 8 or 24 hours, or to hydrogen peroxide (300 µM) for 2 or 24 hours. Sham-treated cells and riebeckite, a non-fibrous mineral, served as controls. After either exposure, the authors reported more c-jun mRNA than in the controls, mainly in the cytoplasm of small groups of cells. This increase was seen in both early apoptotic and non-apoptotic cells, but the increase was not seen in late, fragmented apoptotic cells. Findings were described from images; no counts or statistics were given. The authors concluded the technique is a useful approach for studying gene expression during apoptosis.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory methods paper (cell culture experiment) describing a triple fluorescence labeling technique
• Who was studied: rat pleural mesothelial (RPM) cells isolated from adult Fisher 344 rats and grown in culture, used between passages 5 and 12; University of Vermont laboratory
• Exposure: crocidolite asbestos (5.0 µg/cm2) for 8 or 24 h, or hydrogen peroxide (300 µM) for 2 or 24 h; riebeckite (5 µg/cm2), a non-fibrous, non-cytotoxic mineral chemically similar to crocidolite, used as a negative particle control, plus sham controls
• Main results:
  • An increase in c-jun mRNA was seen in small populations of RPM cells after treatment with crocidolite asbestos or hydrogen peroxide, located mainly in the cytoplasm of clusters or small groups of cells
  • Compared with sham and riebeckite controls, increased c-jun expression in asbestos- or hydrogen peroxide-exposed cells was seen in the cytoplasm of both early TUNEL-positive (apoptotic) and non-apoptotic RPM cells
  • Elevated c-jun expression was lacking in later (fragmented) stages of apoptosis; the authors took this to suggest increased expression early in the apoptotic process
  • Performing TUNEL and in situ hybridization one after the other did not reduce the intensity of the TUNEL signal
  • In control preparations (RNase-pretreated cells or cells hybridized with the sense control probe), increased proto-oncogene expression was absent
• The authors' conclusion: The authors concluded that the technique is a useful in situ approach for evaluating the role of proto-oncogene expression in apoptosis with confocal scanning laser microscopy
• Limitations the authors state:
  • In strongly labeled TUNEL-positive cells, there was a small degree of bleed-through of the APC fluorescent signal; the authors stated it was easily distinguished and did not interfere with interpretation

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Apoptosis, Mesothelial Cells, Crocidolite, Monoclonal.

Related studies

• Multifluorescence labeling techniques and confocal laser scanning microscopy on lung tissue — M. Stern, D.J. Taatjes, et al. (2006)
• Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos — J.L. Goldberg, C.L. Zanella, et al. (1997)
• Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers — C.R. Timblin, G.D. Guthrie, et al. (1998)
• First report of probe based confocal laser endomicroscopy during medical thoracoscopy — O. Bonhomme, B. Duysinx, et al. (2019)
• Apoptosis is observed in mesothelial cells after exposure to crocidolite asbestos — K.A. BéruBé, T.R. Quinlan, et al. (1996)
• Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis — C.L. Zanella, C.R. Timblin, et al. (1999)

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