Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

Coulson, Patricia B.; Bishop, Asa Orin; Lenarduzzi, Roberto

doi:10.1177/25.10.72097

Cited by 49 publications

(10 citation statements)

References 5 publications

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“…As was documented before in various cell systems including stimulated lymphocytes (9)(10)(11)(12)17) and confirmed here (Table 1), the F>600 value represents mostly cellular RNA whereas F53o is a measure of the DNA content per cell (9)(10)(11)(12)18 (3)(4)(5). By using flow cytometry, we observe a similar variation; whereas some lymphocytes double their DNA within 5 hr, others replicate DNA at rates up to 5 times slower (Fig.…”

Section: Resultssupporting

confidence: 87%

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

Darżynkiewicz¹,

Evenson²,

Staiano‐Coico³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACr A flow cytometric technique for simultaneous measurements of RNA and DNA in individual cells has been applied to correlate the content of cellular RNA with the rate of progression of cells through the S phase. Human peripheral lymphocytes stimulated with phytohemagglutinin were blocked at the G1/S phase boundary by hydroxyurea or 5-fluorodeoxyuridine treatment. Cells in the G1 phase as well as cells blocked at the G1/S phase boundary showed high heterogeneity with respect to stainable RNA content. After release from the block, the cells traversed the S phase at rates proportional to the quantity of stainable RNA per cell. Cells with the highest RNA content completed DNA replication 5 hr after release from the block; the cells with minimal RNA traversed the S phase at one-fifth of this rate. The large intercellular variation in stainable RNA and length of the S phase may be due to functional heterogeneity in the lymphocyte population. Our results suggest a correlation between the number of ribosomes and the rate of DNA replication in lymphocytes. Peripheral blood lymphocytes are quiescent (Go) cells that may be stimulated in culture by mitogens to enter the cell cycle (for reviews, see refs. 1 and 2). After stimulation, lymphocytes asynchronously leave the Go state, traverse the G1 phase, and then initiate DNA replication (1, 2). In contrast to most eukaryotic cell types, which have an S period of relatively constant duration (6-9 hr), stimulated lymphocytes are characterized by a large intercellular variation in the length of the S phase (3-5). Duration of the S phase in these cells has been shown to vary from 6 to as much as 30 hr (3-6), with mean values ranging between 9 and 12 hr (7,8 Lymphocytes. Cell collection, separation, and culture methods were described in detail (10,12,13). Briefly, the blood was collected by venipuncture of healthy volunteers, and the leukocytes were separated by gradient centrifugation on Ficoli-Isopaque (Lymphoprep; Nyegaardo, Oslo, Norway). The mononuclear cells were suspended in Eagle's basal medium containing 15% fetal calf serum and subcultured on plastic dishes to remove most of the monocytes (13). The nonadhering cells were then suspended in Eagle's basal medium containing penicillin, streptomycin, and 15% fetal calf serum and incubated at 370C in the presence of phytohemagglutinin (12, 13). To synchronize the cells at the GI/S phase border, the cultures incubated with phytohemagglutinin for 24 hr were treated with 1 mM hydroxyurea (Sigma) or 0.1 tM 5-fluorodeoxyuridine (Sigma) for the next 18-24 hr.Cell Staining. Portions (0.2 ml) of suspension cultures, containing approximately 1-4 X 105 cells, were mixed with 0.4 ml of 0.08 M HCl/0.15 M NaCl/0.1% Triton X-100 (Sigma) at 4VC. The cells were stained 30 sec later by the addition of 1.2 ml of a solution containing 0.2 M Na2HP04/0.1 M citric acid buffer (pH 6.0), 1 mM Na EDTA, 0.15 M NaCl, and AO (chromatographically purified; Polysciences, Inc., Warrington, PA) at 6 ,tg/ml. Considering cell number per sample, the aver...

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Section: Resultssupporting

confidence: 87%

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

Darżynkiewicz¹,

Evenson²,

Staiano‐Coico³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Under these conditions, interaction of the dye with DNA results in green fluorescence with maximal emission at 530 nm and interaction with RNA gives red metachromasia at 640 nm (2). The intensity of green fluorescence was linearly proportional to the content of DNA per cell within the range 2-93 pg (9), and the intensity of red fluorescence correlated with RNA content (r = 0.93, see ref. 10).…”

mentioning

confidence: 92%

Subcompartments of the G1 phase of cell cycle detected by flow cytometry.

Darżynkiewicz¹,

Sharpless²,

Staiano‐Coico³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

183

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Cellular DNA and RNA were simultaneously quantitated in individual cells of cycling and quiescent populations by flow cytometry. Based on differences in RNA content, two distinct subcompartments, GIA and GIB, were recognized in the G1 phase of exponentially growing cells. After mitosis, cells reside in the low-RNA, GIA compartment from which they cannot enter the S phase directly. An increase in RNA above a critical level is required for G1 cells to be able to initiate DNA replication; G1 cells with RNA values above this level are classified as GIB. Cell transition from GIA to GIB and from GIB to S was analyzed in a stathmokinetic experiment on L1210 cells with a doubling time of 10 hr. The half-time of cell residence in the indeterminate state of G1 was found to be 1 hr, and the duration of the deterministic portion of G1 phase was 2 hr. described (8). Cells were studied either during the exponential phase of growth or during quiescence induced by serum deprivation, prolonged maintenance until confluence, or addition of sodium n-butyrate, as described in the figure legends. Other details related to the cell system and preparation of cells for measurements by flow cytometry are given elsewhere (2-6, 8).Cell Staining and Fluorescence Measurements. A detailed description of the technique is given elsewhere (2, 3). Briefly, cells made permeable by Triton X-100 (Sigma) were stained with chromatographically purified acridine orange (Polysciences Inc., Warrington, PA) at a final dye concentration of 13 MIM and dye/DNA-P molar ratio >2 in the presence of 1 mM Na EDTA. Under these conditions, interaction of the dye with DNA results in green fluorescence with maximal emission at 530 nm and interaction with RNA gives red metachromasia at 640 nm (2). The intensity of green fluorescence was linearly proportional to the content of DNA per cell within the range 2-93 pg (9) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…Fixed cells were washed two times with ice-cold PBS buffer and were kept in PBS at 4°C. DNA in cells was stained with propidium diiodide (PI) (Sigma-Aldrich), dissolved in PBS (final concentration 2.5 mg/ mL suspension; Coulson et al, 1977;Coulson and Tyndall, 1978), and simultaneously treated with RNase A (final concentration 10 mg/mL) for 3 h at 4°C. Prior to cytometric analysis, cells were washed once with ice-cold PBS.…”

Section: Dna Content and Ploidymentioning

confidence: 99%

Systems-Wide Analysis of Acclimation Responses to Long-Term Heat Stress and Recovery in the Photosynthetic Model OrganismChlamydomonas reinhardtii

et al. 2014

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We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42°C for 24 h and back to 25°C for ‡8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions.

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Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

Cited by 49 publications

References 5 publications

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

Subcompartments of the G1 phase of cell cycle detected by flow cytometry.

Systems-Wide Analysis of Acclimation Responses to Long-Term Heat Stress and Recovery in the Photosynthetic Model OrganismChlamydomonas reinhardtii

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