Rapid, Simultaneous Measurement of Dna, Protein, and Cell Volume in Single Cells From Large Mammalian Cell Populations

Crissman, Harry A.; Steinkamp, John A.

doi:10.1083/jcb.59.3.766

Cited by 513 publications

(215 citation statements)

References 17 publications

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“…For quantitation of DNA cell pellets were fured and stained for DNA with propidium iodide (PI) as previously described (10,24). For measurements of both T antigen and DNA, fmed cells were fxst stained for T antigen and then by the PI protocol.…”

Section: Methodsmentioning

confidence: 99%

SV40‐Infected muntjac cells: Cell cycle kinetics, cell ploidy and T antigen concentration

Gershey

1980

Cytometry

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Muntjac cells in which the SV40 virus neither readily causes transformation nor replicates were used to study the effect of SV40 infection on cell ploidy and the influence of ploidy on the concentration of T antigen, which is required for the initiation of viral DNA synthesis. Both the DNA content, as measured by flow microfluorometry of propidium iodide-DNA fluorescence, and the average number of chromosomes per cell indicated that infection with SV40 did not alter the ploidy of the host cell. SV40 infection had no effect on the ploidy distribution of muntjac cells. After immunofluorescence staining with anti-T serum and fluorosceinlabeled anti-yG, infected and uninfected cultures were compared. In uninfected cells incubated with a 1:20 dilution of anti-T serum no fluorescence could be observed by fluorescence microscopy, but when examined by flow microfluorometry, fluorescence was detected after staining with as little as 1000-fold diluted antiserum. Determination of the amount of T antigen and DNA content in the same cell by simultaneous measurement of fluorescein isothiocyanate-conjugate and propidium iodide fluorescence, indicated that the cellular concentration of T antigen did not vary with the ploidy of the genome or the number of nuclei per cell. These results suggest that gene dosage is not a factor which determines the permissive environment for SV40 replication. Key terms: Flow microfluorometry, SV40, virus-host cell interactionsWe have previously reported that a viral regulatory protein, SV40 T antigen, binds to the chromosomes of muntjac cells (13). It is known that SV40 T antigen is required for the reinitiation of each round of viral DNA replication (32, 50), but the functional significance of its binding to the chromosomes of cells in which SV40 does not replicate remains unclear. By comparison with the chromosome banding pattern visualized by quinacrine staining, it appears that T antigen is binding near AT-rich regions of the DNA (14). Such bands have been associated with units of cellular DNA replication (33). AT clusters are common to sequences at or near the origins for DNA replication as has been shown by nucleotide sequencing of papovaviruses (15,22,49), adenoviruses (52), parvoviruses (2) and bacteriophages (31,39,43).

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Section: Methodsmentioning

confidence: 99%

SV40‐Infected muntjac cells: Cell cycle kinetics, cell ploidy and T antigen concentration

Gershey

1980

Cytometry

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“…For many decades, PI has been widely used as a dye for measuring relative DNA content in single parameter flow cytometry (5,10,11). Since many investigators became interested in multiparameter analysis, PI has also been used in combination with antibody labeling (16).…”

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confidence: 99%

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

1994

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A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper.A subequilibrium concentration of 1 pM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In humanand mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation.Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. 0 1994 Wiley-Liss, Inc.

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“…Propidium iodide (PI) was first described as a quantitative DNA stain by Crissman and Steinkamp (2). Since then, it has appeared to be an easy to handle dye with high specificity for DNA.…”

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The fluorescence intensity of propidium iodide bound to DNA depends on the concentration of sodium chloride

Martens¹,

Engh²,

Hagenbeek³

1981

Cytometry

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While performing DNA analysis with propidium iodide using the FACS-I1 cell sorter, it was noted that the fluorescence intensity was not constant during the measurements. W h e n different sheath and sample fluids were used, the fluorescence intensity of a given cell population increased or decreased during the m e a s u r e m e n t , depending on the concentration of sodium chloride in the fluids. By selecting the appropriate sheath and sample fluid combination, the drift in fluorescence intensity can be avoided.Key terms: Propidium iodide, signal stability, cell cycle, DNA analysis Propidium iodide (PI) was first described as a quantitative DNA stain by Crissman and Steinkamp (2). Since then, it has appeared to be an easy to handle dye with high specificity for DNA.A rapid method for PI staining under hypotonic conditions was described by Krishan (3) and modified by Vindelov (6) to allow DNA determinations in needle aspirates of solid tumors. Such a staining method is useful for scheduling chemotherapy in tumor-bearing patients. Cell cycle phase specific drugs should be given at the time when optimal results of the treatment can be expected. Especially for combination chemotherapy or for monitoring recruitment of cells into cycle rapidly DNA analysis is indispensable. Its usefulness has been shown by many authors (1,5). For this purpose, DNA histograms before and after treatment must be compared. Differences in the fluorescence intensity must be related to DNA concentration. Therefore, complete stability is required during the measurements. Even then, internal standards may prove to be necessary. In flow cytometry (FCM), the sample is injected in a fluid stream-the sheath fluid. Although the buffer containing the sample is usually carefully chosen, little attention has been paid to the composition of the sheath fluid.In this study, it was found that the composition of the sample and sheath fluids may influence the intensity and the stability of the PI fluorescence. Materials and MethodsCell Suspension and Fixation: The experiments were performed with thymus cells of the Brown Norway inbred rat strain. DNA Staining: The cells were taken from the pellet and stained with propidium iodide at 50 mg/liter (CalBiochem, San Diego, CA) in the presence of 0.1% sodium citrate. The cell concentration during the staining procedure was adjusted to 3 X 10' cells/ml. Before measurements were performed, the cells were washed two times (10 min at 400 g at 4°C) and resuspended in either distilled water or in a solution of the desired sodium chloride osmotarity.Fluorescence Measurements: The cells were analyzed with a FACS-I1 cell sorter (Becton Dickinson FACS Systems, Sunnyvale, CA). The system operates with an Argon laser of which the 488 nm line was used. Fluorescence above 620 nm was measured. The cells were transported to the nozzle with increased pressure ("boosting"). As soon as the first cells arrived, the measurements were started. ResultsThe instability of the PI measurement was tested under two conditions. The results a...

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Rapid, Simultaneous Measurement of Dna, Protein, and Cell Volume in Single Cells From Large Mammalian Cell Populations

Cited by 513 publications

References 17 publications

SV40‐Infected muntjac cells: Cell cycle kinetics, cell ploidy and T antigen concentration

SV40‐Infected muntjac cells: Cell cycle kinetics, cell ploidy and T antigen concentration

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

The fluorescence intensity of propidium iodide bound to DNA depends on the concentration of sodium chloride

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