Practical Flow Cytometry

Shapiro, Howard M.

doi:10.1002/0471722731

Cited by 1,153 publications

(791 citation statements)

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Section: Discussionmentioning

confidence: 99%

“…Unfortunately, this is not always true for whole range of signals, and might be nonideal for small and very HFR intensity. In the case of RET, which falls in the middle of signal range an ideal behavior typically holds, allowing a quantitative interpretation of acquired signals (20,21).The obtained degradation rate is difficult to explain mechanistically. It is evident that RNA is degraded throughout the maturation of RET in blood with the rate that depends on RNA content (Fig.…”

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confidence: 99%

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Method of determination of the reticulocyte age distribution from flow cytometry count by a structured‐population model

Wiczling

Krzyzanski

2007

Cytometry Pt A

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Background: A flow cytometry count of reticulocytes (RET) provides information about distribution of signal emitted by reticulocyte RNA. A new method of determination of the RNA degradation rate in RET is provided. This technique allows one to determinate the age distribution of RET. Methods: The method is based on a series of flow cytometry counts of cultured RET. From those counts, the changes of signal distribution of RET over time are obtained. The RNA degradation rate of individual RET is then resolved based on changes in the signal distribution of the whole population of cells. The obtained relation between signal and age allows obtaining a RET age density that can be used to characterize RET age distribution and age dependence of processes that control the population dynamics. Results: The total maturation time of RET in rats is 3 days. The median time that a homeostatic RET spends in blood or before it becomes a mature RBC is about 0.6 days, whereas a stress RET needs 0.8 days. Conclusions:The proposed method provides means for studies in vivo RET dynamics using age-structured models. Key terms: reticulocytes; structured population model; RNA degradation; flow cytometry Reticulocytes (RET) are immature red blood cells containing one or more filaments or granules of ribosomal RNA. They are formed during mammalian erythropoiesis through the nuclear exclusion from last stage erythroblasts. Then, after a period of maturation in bone marrow, RET are released into the blood circulation and undergo further differentiation to mature erythrocytes. During that period, RET change from large and motile cells to small, biconcave, discoid cells (1-3). In rats, the RET residence time in the bone marrow is about 17-24 h (4,5). The in vivo disappearance of RET population from blood is linear and takes 30 h (5). In vitro culture studies showed that the RET lifespan in rats is about 2-3 days (5-7). The appearance of young RET in blood, called stress RET, gives an insight into the response of bone marrow to endogenous erythropoietin stimulation, treatment with recombinant human erythropoietin, or other erythropoietic stimuli. Hence, it is used as a relatively fast marker of undergoing erythropoiesis (2). The time stress RET spent in bone marrow can decrease to 6.5 h in rats.The lack of capacity for RNA synthesis in RET causes a progressive decrease in cell content of RNA and is one of the characteristics of maturation of RET and consequently provides means of assessing the RET maturation time. In vitro culture data from rabbits show a linear decline of the RNA content at a rate of about 4% per hour that has been determined over a limited time (8). In rats, an exponential decline with half-life about 24 h was found (9). An analogous decline with half-life of 43 h was reported in humans (10).Enumeration of peripheral RET is primarily done by means of automated counting. For flow cytometry measurements, the RET RNA is labeled with a fluorescence dye and the fluorescence of the dye-RNA complex is recorded (11). In general, youn...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Method of determination of the reticulocyte age distribution from flow cytometry count by a structured‐population model

Wiczling

Krzyzanski

2007

Cytometry Pt A

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“…Unfortunately, the quantum yield highly depends on the environment, so it is difficult to tell the value of Q D for a given donor dye under measurement conditions. However, it should be noted that R 0 is mostly sensitive to Q D in the range of 0.0 to 0.3 (28), which is the characteristic range for cyanine dyes, whereas the Alexa dyes have quantum yield values about or higher than 0.3 in our experience (data not shown); for further informa- tion, please see Shapiro (32), pp. 336-338.…”

Section: Establishing Factors That Influence Fcet Measurementsmentioning

confidence: 84%

“…Unfortunately, we could not test flow cytometers other than those from Becton Dickinson, but we have assessed other cytometers from different manufacturers available on the market; for a thorough list, see chapter 8 in Shapiro (32). We found that the FC500 (Beckman Coulter, Fullerton, CA, USA) and the CyAn (DakoCytomation, Fort Collins, Colorado, USA) have characteristics similar to the FACSCalibur; they share the same light sources, detection filters, and cuvette system, so the results presented in this report are presumably valid for these two instruments and for two other instruments from Becton Dickinson, the LSR II and FACSAria.…”

Section: Discussionmentioning

confidence: 99%

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements

et al. 2005

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Background: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. Methods: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and b2-microglobulin with antibodies conjugated with fluorescein-and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. Results: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye-and instrument-

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“…Recently flow cytometric (FCM) detection and quantification of virus particles has been demonstrated (7,(9)(10)(11)(12)(13)(14)(15). The use of FCM requires staining the virus with a fluorescent dye because viruses are generally too small to efficiently scatter light (12).…”

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confidence: 99%

Design and characterization of a compact dual channel virus counter

2005

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Background: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the ''gold standard.'' Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. Methods: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels.

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Practical Flow Cytometry

Cited by 1,153 publications

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Method of determination of the reticulocyte age distribution from flow cytometry count by a structured‐population model

Method of determination of the reticulocyte age distribution from flow cytometry count by a structured‐population model

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements

Design and characterization of a compact dual channel virus counter

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