Buoyant density separation of cells. I. The buoyant distribution of guinea pig bone marrow cells.

Leif, Robert C.; Smith, Sheryl S.; Dunlap, Larry A.; Leif, S. B.

doi:10.1177/23.5.1127223

Cited by 10 publications

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“…AchE is an enzyme which has been shown to be relatively specific for the megakaryocytic lineage in rodents, and can be identified in small cells which are unrecognizable as megakaryocytes by standard morphologic features (Jackson, 1973;Long and Henry, 1979;Long and Williams, 1981). Techniques for the separation and partial isolation of these classes of megakaryocytes have been described (Nakeff and Maat, 1974;Leif et al, 1975; 0 1985 ALAN R. LISS, INC Levine and Fedorko, 1976;Nakeff and Floeh, 1976;Pretlow and Stinson, 1976;Nachman et al, 1977;Sitar et al, 1977;Rabellino et al, 1979;Levine, 1980;Worthington and Nakeff, 1981;Williams et al, 1981;Long et al, 1982;Sitar, 1984). Rabellino et al (1979) found that mature human megakaryocytes are found at the top of discontinuous Percoll gradients at low density (less than 1.050 g/cm3).…”

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Separation of murine megakaryocytes and their progenitors on continuous gradients of percoll

Ishibashi

Burstein

1985

Journal Cellular Physiology

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Murine bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocyte lineage. Eighty-seven percent of the recovered megakaryocytes were found in fractions of density less than 1.058 g/cm3, with 63% of these cells found between 1.020 and 1.036 g/cm3. When megakaryocytes were classified according to size, 92% of the large (greater than or equal to 18 micron) acetylcholinesterase (AchE) positive cells were found in the least dense fractions (1.016-1.039 g/cm3), whereas 86% of the small (less than or equal to 10.6 micron) AchE positive cells were found in fractions of higher density (1.039-1.078 g/cm3). The distribution of enzymatic AchE activity of the separated fractions corresponded to the location of the histochemically positive cells. When ploidy measurements were made of various fractions, most of the high ploidy (32N and 64N) cells were found at low density (1.028-1.036 g/cm3), whereas no cells greater than 4N were found at density greater than 1.071 g/cm3. Thus, large AchE positive cells and the cells of highest ploidy were found at lower densities of Percoll, while small AchE positive cells and cells of low ploidy were found at higher densities. An exception to this inverse relationship was found in fractions of lowest density (less than 1.030 g/cm3) where an anomalous distribution of size and ploidy was found. The majority of megakaryocytic colony-forming cells (CFU-MK) were found at high density, as were the granulocyte-macrophage colony-forming cells (CFU-GM; approximately 1.074 g/cm3). The density distribution of the incorporation of tritiated thymidine into liquid marrow cultures was concordant with the high density distribution of colony-forming cells. The data show that megakaryocytic maturity and Percoll density varies inversely and that fractionation of marrow on continuous Percoll gradients may be a useful method for the separation and/or enrichment of megakaryocytes at different stages of differentiation.

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Separation of murine megakaryocytes and their progenitors on continuous gradients of percoll

Ishibashi

Burstein

1985

Journal Cellular Physiology

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Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes

Leif

Schwartz

Rodriguez³

et al. 1985

Cytometry

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Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.Key terms: Coulter electronic cell volume, opacity, erythrocytes, impedance, radio frequency, buoyant density, bovine serum albumin (BSA), density gradient centrifugation Leif and co-workers (9,131 proposed that automated cell analysis should be based on the assumption that there are many descriptors available that permit the easy identification of each type of cell present in a heterodisperse population. However, the use of a combination of two impedance measurements (13,5) together with multidimensional fluorescence and light scattering data on a cell-by-cell basis should allow adequate quantification of cellular parameters for specific identification of cells, For flow analysis to maintain a high level of accuracy, it will be necessary to include redundant information by increasing the dimensionality of data in order to prevent errors.The Coulter volume measurement, which was one of the first parameters to be used for the automated counting and analysis of biological cells (2,3), is an impedance measurement at zero frequency (dc) or a measure of resistance. Besides the abovementioned dc resistance (electronic cell volume) measurements, it is possible to perform simultaneously ac impedance measurements. An instrument for this purpose has been developed by Coulter and Hogg (4). The highest resolution in a twodimensional distribution is achieved by producing orthogonal parameters. In the present case, this was accomplished by dividing the radio frequency ac impedance by the dc resistance, which is defined as the opacity.Hoffman and Britt (6) developed a flow-system technique that simultaneously detected the dc Coulter volume and ac impedance. As pointed out by the authors, the use of the low ac frequency of 1 MHz resulted in "a high correlation between the ac and dc parameters for CHO cells." These authors performed their measurements at the reactance maximum in order to maximize the difference between cell types (Britt, personal communication) rather than at the higher frequency range.The ac impedance changes slowly with frequency above approximately 20 MHz, where, according to the bulk measurements of erythrocyte suspensions by Jenin et al.(71, the conductance of intact erythrocytes plateaus. According to these authors, the erythrocyte membrane appears to be practically short-circuited at frequencies above 50 MHz.The purpose of this study was to determine the relationship of the electrical parameters of Co...

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A short history of the initial application of anti–5‐BrdU to the detection and measurement of S phase

Leif

Stein²,

Zucker

2004

Cytometry Pt A

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Because this article is a bit of history rather than a scientific paper, we will depart from the formality of a journal article. This article has two purposes. The first is to give credit to two deceased individuals, Albert Castro, M.D., and Howard Gratzner, Ph.D. (1). Castro was the key person who was able to produce a polyclonal antibody that was specific for bromodeoxyuridine (BrdU). For 10 years, Gratzner took the lead in converting an idea into a functional assay that affected DNA analysis and led to the development of many other assays with similar methodologies. The second is to inform young scientists that (a) inventions or creations have utility in fields other than the purpose for which they were created; (b) it is possible to do useful work in an institution that certainly would not have been classified as one of the best; and (c) the presence of talented people who are willing to collaborate is invaluable.Scientifically, Gratzner is best known for the development of the first monoclonal antibody (mAb) to BrdU and iododeoxyuridine. This work was the culmination of previous efforts with Robert Leif to develop polyclonal antibodies, which detected BrdU with variable specificity. Collaborations with scientists at the Lawrence Livermore Laboratory encouraged the development of a widely used flow cytometric test for the simultaneous measurements of DNA and BrdU. The use of mAbs to halogenated pyrimidines was a significant scientific advance and currently is an integral tool for the analysis by flow and digital microscopy of the cell cycle.On a personal level, Gratzner's enthusiasm for science and his intelligence, curiosity, loyal interactions with fellow collaborators, and friendship are sorely missed by all who knew him. He was genuinely a warm and nice person who was liked by almost everyone with whom he came in contact (1).Castro was a jovial man who interacted well with his collaborators. He was generous and very knowledgeable. He ran an immunodiagnostics laboratory at the universities of Oregon and Miami and was an expert in the production of antibodies in animals. His warm smile, generous giving of his knowledge, common sense, enthusiasm for scientific research, and ability to get the job done were essential to this project.Because this is a story of events that happened approximately 30 years ago and the existing records are limited, the accuracy of this article is limited by our memory and those of our colleagues who have kindly helped with this project. Of course, this is our version of the events.Cell division and DNA replication are fundamental to biology and specifically to the biology of cancer. This article describes how a simple procedure for the detection and quantitation of DNA synthesis was developed. The precise determination of when S phase occurs in the cell cycle is of use to maximize the selective killing of tumor cells with cycle-specific chemotherapeutic drugs. Of greater significance, studies of the control of the cell cycle, including its detour into apoptosis, can provide extre...

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Buoyant density separation of cells. I. The buoyant distribution of guinea pig bone marrow cells.

Cited by 10 publications

References 0 publications

Separation of murine megakaryocytes and their progenitors on continuous gradients of percoll

Separation of murine megakaryocytes and their progenitors on continuous gradients of percoll

Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes

A short history of the initial application of anti–5‐BrdU to the detection and measurement of S phase

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