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Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.

ABSTRACT: Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.

SUBMITTER: Osypiw JC

PROVIDER: S-EPMC1137536 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Project description:In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.

Project description:BackgroundPlasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates.MethodsPlasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28.ResultsAfter stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28.ConclusionThe combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium.

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Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.

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