Flow cytometry of human reticulocytes based on RNA fluorescence

Tanke, Hans J.; Nieuwenhuis, I. A. B.; Koper, Ger J. M.; Slats, J.; Ploem, J. S.

doi:10.1002/cyto.990010503

Cited by 82 publications

(48 citation statements)

References 8 publications

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“…The peak fluorescence intensity did shift slightly depending on the concentration of PY (not shown). The characteristics of PY staining have been described previously by Tanke et al (28). The MG-PY staining was found to be stable between pH 5-7 and was maximal after 30 min.…”

Section: Methodsmentioning

confidence: 52%

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Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green

et al. 1982

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Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3-to &fold reduction in the fluorescence intensity of intracellular MG-PY.Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24,48, and 72 hr of cell culture were monitored, The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular DNA whereas increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.Key terms: Pyronin Y, methyl green, flow cytometry, RNA Over the last decade the automated identification and classification of cellular subpopulations has developed extensively using flow cytometric (FCM) instrumentation. This instrumentation has been particularly useful in the rapid discrimi-'

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Section: Methodsmentioning

confidence: 52%

“…Tanke et al (28) introduced the use of pyronin Y (PY) staining for the quantitation of RNA per cell by flow cytometry (FCM). They used the fluorescence from PY to quantitate reticulocytes based on RNA content.…”

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confidence: 99%

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green

et al. 1982

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“…This is based on RNA fluorescence by use of pyronine-Y staining and seems to be a promising method for the study of anemias and other red cell diseases (193). Other new and interesting methods are the detection and sorting of sickle hemoglobin-containing erythrocytes in human blood by use of fluorescent labelled antibodies (145), as well as the analysis and sorting of plasmodiumand trypanosoma-infected red blood cells by use of parasite DNA staining with e.g., the fluorescent dye Hoechst 33258 (105,106,147).…”

Section: Blood and Bone Marrow Cellsmentioning

confidence: 99%

Clinical application of flow cytometry: A review

1981

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A survey of the use of flow cytometry for clinical purposes is given. In the last decade the main clinical application of this technique has been measurements of cellular DNA content for estimation of cell cycle distribution and ploidy studies. A large body of data is now available on the presence of aneuploidy in different malignant diseases. By measurements with high resolution, the demonstration of abnormal cellular DNA content in several types of t u m o r s can be of definite diagnostic value when combined with conventional diagnostic procedures. The prognostic significance of different types of DNA aberrations is so f a r not established. Attempts to monitor cancer treatment by studying altered cell cycle distribution have not been successful, although s o m e applications are of potential value. The main reasons f o r this are the complexity of t u m o r tissue as well as difficulties with interpretation of altered cell cycle distribution caused by drug combinations. For further progress in this field more emphasis on other cell constituents than DNA measured b y flow cytometry is desirable, either as single or as multiparameter measurements.

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“…Although invaluable and adequate for many studies, this technique limits the number of samples that can be evaluated within an experiment as well as the number of cells counted per sample and thereby the sophistication with which a problem may be approached. The use of laser-based fluorescence flow cytometry in blood cell identification has been explored (1,12,23). Reports are lirxiiled to the counting and classification of WBC (1,12) and reticulocytes (23).…”

mentioning

confidence: 99%

“…The use of laser-based fluorescence flow cytometry in blood cell identification has been explored (1,12,23). Reports are lirxiiled to the counting and classification of WBC (1,12) and reticulocytes (23). Applications of flow cytometry in malaria research have also been published (5,7,8,15).…”

mentioning

confidence: 99%

Analysis of malaria parasite‐infected blood by flow cytometry

1983

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The use of flow cytometry in the quantitative analysis of blood from mice infected with Plasmodium vinckei has been studied. Several fluorescent dyes responsive to cell membrane potential were screened and one dye, 3,3'-dimethyloxacarbocyanine (I)ioc1(3)), was chosen for further study. Mature red blood cells (mRBC), immature RBC (imRBC), and parasitized RBC (pRBC) could be recognized and counted in the flow cytometer. When infected blood was separated on a Percoll gradient and fractions analyzed by flow cytometry using DiOC1(3), distinct populations of pRBC were recognized, the frequency of which varied with density. These subpopulations could not be correlated with distinct morphologic stages but varied with the size or age of the growing parasite. Methods combining the use of DiOC1(3) with a DNA specificdye, Hoechst 33342, are discussed as an approach to more complete analysis of the blood of malaria-infected animals.

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Flow cytometry of human reticulocytes based on RNA fluorescence

Cited by 82 publications

References 8 publications

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green

Clinical application of flow cytometry: A review

Analysis of malaria parasite‐infected blood by flow cytometry

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