Correlated Measurements of DNA, RNA, and Protein in Individual Cells by Flow Cytometry

Crissman, Harry A.; Darżynkiewicz, Zbigniew; Tobey, Robert A.; Steinkamp, John A.

doi:10.1126/science.2408339

Cited by 140 publications

(102 citation statements)

References 20 publications

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“…In both AIF þ /y and AIF À/y ES cells, RNA degradation was abolished by addition of the pan-caspase inhibitor Z-VAD-fmk. This result was obtained using two distinct methods for the evaluation of total cellular RNA content, namely RNA extraction followed by horizontal agarose gel electrophoresis (Figure 4b) or staining with the RNA probe pyronin Y (Crissman et al, 1985;Halicka et al, 2000), followed by cytofluorometric evaluation of the per-cell RNA content (Figure 4c). Thus, in accord with the literature (Rutjes et al, 1999;King et al, 2000;Asselbergs and Widmer, 2003), apoptotic RNA degradation is caspase-dependent, and, as shown in Figure 3b and c, AIF-independent.…”

Section: Resultsmentioning

confidence: 98%

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Physical interaction of apoptosis-inducing factor with DNA and RNA

et al. 2005

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Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which upon apoptosis induction translocates to the nucleus where it interacts with DNA by virtue of positive charges clustered on the AIF surface. Here we show that the AIF interactome, as determined by mass spectroscopy, contains a large panel of ribonucleoproteins, which apparently bind to AIF through the RNA moiety. However, AIF is devoid of any detectable RNAse activity both in vitro and in vivo. Recombinant AIF can directly bind to DNA as well as to RNA. This binding can be visualized by electron microscopy, revealing that AIF can condense DNA, showing a preferential binding to singlestranded over double-stranded DNA. AIF also binds and aggregates single-stranded and structured RNA in vitro. Single-stranded poly A, poly G and poly C, as well doublestranded A/T and G/C RNA competed with DNA for AIF binding with a similar efficiency, thus corroborating a computer-calculated molecular model in which the binding site within AIF is the same for distinct nucleic acid species, without a clear sequence specificity. Among the preferred electron donors and acceptors of AIF, nicotine adenine dinucleotide phosphate (NADP) was particularly efficient in enhancing the generation of higher-order AIF/ DNA and AIF/RNA complexes. Altogether, these data support a model in which a direct interaction of AIF contributes to the compaction of nucleic acids within apoptotic cells.

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Section: Resultsmentioning

confidence: 98%

“…To assess RNA content, ES cells were trypsinized and stained with pyronin Y (1 mg/ml, Sigma) and incubated at 371C for 20 min (Crissman et al, 1985;Halicka et al, 2000). Stained cells were then analysed in a FACS Vantage s (Becton & Dickinson).…”

Section: Cytofluorometrymentioning

confidence: 99%

Physical interaction of apoptosis-inducing factor with DNA and RNA

et al. 2005

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“…In some experiments a three-laser flow cytometer was used; the instrument is described elsewhere (4,27 Figure 1 shows CHO cells stained with 1 mM PY, observed by bright field microscopy. Localization of the colored product in the cytoplasm and nucleoli and its absence in nucleoplasm are typical of the distribution of RNA.…”

Section: Methodsmentioning

confidence: 99%

“…(4). This allows measurement of small quantities of RNA under conditions of high DNA/RNA ratios (e.g., as in isolated nuclei) without a problem of spectrum overlap from DNA.…”

Section: (83)mentioning

confidence: 99%

Application of pyronin Y(G) in cytochemistry of nucleic acids

et al. 1987

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Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y (PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation.The products of PY interaction with doublestranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry.PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 pM, the RNA stainability decreases, perhapsIn an accompanying paper (171, we describe biophysical and biochemical studies on pyronin Y (PY) and interactions of this dye with natural and synthetic nucleic acids in solutions. Several observations from these studies could be of importance for the qualitative and quantitative application of PY, both as an absorption dye for light microscopy (2,191 and as a fluorochrome (4,23,26,28). In the present paper, we demonstrate that the biophysical properties of the dye complexes with nucleic acids observed in solutions (17) are relevant in cytochemistry. The combined biophysical and cytochemical approach as described in this and the accompanying paper (17) makes it possible to better understand the complexity of the dye-nucleic acid interactions and define optimal conditions for use of this dye as a specific cytochemical probe of nucleic acids. MATERIALS AND METHODSChinese hamster cells (CHO, originally obtained from Dr. T.T. Puck) were maintained in exponential growth due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in 6 2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.Key terms: RNA content, RNA conformation, nuclear RNA, flow cytometry, CHO cells, mitosis phase either as monolayer cultures, or in suspension culture, free of mycoplasma contamination, in Ham's F-10 medium supplemented with 10% heat-inactivated newborn calf serum, as previously described (6). To obtain populations enriched in mitotic cells, the suspension cultures were treated with 0.1 pg/ml Colcemid (GIBCO, Grand Island, NY) for up to 3 hr; this resulted in a n...

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“…The effects of furan-2-carboxamide derivative on the intracellular metabolism of L. mexicana promasti- gotes were estimated by determining cell cycle and total protein contents and compared to those of pen tamidine and allopurinol (Crissman et al, 1985). Pro mastigotes exposed to this compound were fixed with 70 % methanol, centrifuged at 600 g and sus pended in 1 ml RNase solution (50 pg/ml in PBS) at 37° C for 30 min.…”

Section: Flow Cytometrymentioning

confidence: 99%

In vitroandin vivoantileishmanial activity of 2-amino-4,6-dimethylpyridine derivatives againstLeishmania mexicana

Abdala

Alvarez

Delmas³

et al. 2002

Parasite

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Summary:Leishmania mexicana promastigote and intracellular amastigote growths were inhibited by the water-soluble furan-2-carboxamide issued from the pharmacophore 2-amino-4,6-dimethylpyridine with IC50 values of 69 ± 2 and 89 ± 9 μM, respectively. This compound was also tested against established L. mexicana infection in susceptible BALB/c mice; an intraperitoneal administration of 10 mg/Kg/day during five consecutive days induced a high teduction in the amastigote burden of the poplitea lymph node (81 ± 6.4 %), the spleen (80 ± 1.6 %) and the liver (73 ± 9 %). Approach of the mechanism of antileishmanial activity of this compound, assessed by the flow cytometry, showed a reduction in the protein and DNA synthesis. Finally, an actual increase of the in vitro antileishmanial activity was obtained by replacement of the amidic function by an imidazolidin-2-one moiety. In this new seties, two of the N-substitued derivatives showed IC50 values of 1 3 ± 0.5 and 7 ± 3 μM in intracellular amastigotes constituting new promising compounds for further studies.KEY WORDS : leishmania mexicana, antileishmanial activity, 2-amino-4,6-dimethylpyridine, flow cytometry. Résumé

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Correlated Measurements of DNA, RNA, and Protein in Individual Cells by Flow Cytometry

Cited by 140 publications

References 20 publications

Physical interaction of apoptosis-inducing factor with DNA and RNA

Physical interaction of apoptosis-inducing factor with DNA and RNA

Application of pyronin Y(G) in cytochemistry of nucleic acids

In vitroandin vivoantileishmanial activity of 2-amino-4,6-dimethylpyridine derivatives againstLeishmania mexicana

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