Applications of Percoll In Other Cell Types

Extracted from Cell Separation Media Methodology and Applications, GE Healthcare, 2007

The following tables were compiled to assist the researcher in selecting references most likely to contain relevant information regarding use of Percoll for a particular cell or tissue type.

 

Liver cells
Species Gradient type Tissue type Comments Downstream application Ref. #
human continuous liver Purification of cryo-preserved hepatocytes on Percoll density gradients increased the percentage of viable cells from 55 to 87%. primary cell culture, electron microscopy, viability assay radiolabeled protein synthesis, secretion assay, metabolic studies, toxicological studies 975
rat continuous liver Percoll offered a good way to obtain an enriched population of Kupffer cells. Recovery was 82%, viability 87% and purity 67%. peroxidatic reaction 20
rat continuous liver Percoll gradients were used to isolate hepatocyte plasma membranes and mitochondrial membranes. phase contrast microscopy, cell binding experiments 33
rat continuous liver Rat liver cells furnished subpopulations of parenchymal cells (hepatocytes) having buoyant densities of 1.07 to 1.09 g/ml, and non-parenchymal cells (mostly phagocytosing Kupffer cells) at a density of 1.04 to 1.06 g/ml. cell culture 55
rat NA liver Final preparations contained less than 5% nonviable cells as judged by trypan blue exclusion. cell culture 71
rat continuous liver Percoll gradients were used to franctionate nonparenchymal cells into Kupffer cells, stellate and endothelial cells. light and flourescence microscopy, carboxyesterase and Glutathione- S-transferase (GST) activities 976
rat discontinuous (2-layer) liver Percoll provided a simple, low cost, and rapid method for the isolation, purification and cultivation of rat liver sinusoidal endothelial cells (LEC). electron microscopy, cell culture, trypan blue exclusion 977
rat discontinuous (2-step) liver Percoll gradients were used to separate fat storing cells (FSC) from liver endothelial cells (LEC) and Kupffer cells (KC). cell culture 978
rat continuous liver Following the removal of damaged cells by centrifugation in Percoll, the mean viability of cryo-preserved hepatocytes, tested by trypan blue exclusion, was 88.6% (±1.3%). cell viability and study of xenobiotic metabolism 979
rat continuous liver Percoll was used to remove dead cells from cryopreserved cells. Cell viability was 88 ±1% after the Percoll step. cell viability and study of xenobiotic metabolism 980
rat continuous liver If cryo-preserved cells were purified by a Percoll centrifugation after thawing, the enzyme activities were not significantly different from those of freshly isolated parenchymal cells, and the viability was 86%. Lowry protein assay, cytochrome assay, enzyme assays 981
rat continuous liver Percoll separation yielded cryo-preserved cells with a viability and metabolic capacity not measurably different from freshly isolated cells. protein determination, enzyme assays and metabolism of testosterone and benzo(a) pyrene (BaP) 982
rat discontinuous (2-layer) liver Percoll two-step gradients were used to separate Kupffer cells (KC) and liver endothelial cells (LEC). Preparations of KC were 85 to 92% homogenous while the LEC preparation was at least 95% pure. light microscopy, electron microscopy and peroxidase staining 983
rat discontinuous (5-layer) liver, spleen Percoll gradients were used to separate both spleen and liver cells. Spleen and liver cell viability was over 95%. trypan blue viability assay, cell culture 984
rat continuous liver biopsy Percoll was used for separation of hepatocytes and non-parenchymal cells, as well as subfractionation. cell enumeration using Coulter counter, immunocytochemistry, DNA extraction, Southern blot analysis, assay of marker enzymes and protein in subcellular fractions, electron microscopy 985

 

Leydig cells
Species Gradient type Tissue type Comments Downstream application Ref. #
human continuous testis Percoll-purified Leydig cells were 70 to 80% pure based on staining for 3 beta-hydroxysteroid dehydrogenase. cell culture, stimulation of testosterone production 986
human continuous testis Percoll-purified Leydig cells were 80 to 90% pure as determined by 3 betahydroxysteroid Dehydrogenase staining. immunocytochemical localization of apolipoprotein E (apoE) 987
human discontinuous (4-layer) testis Percoll gradients were used to isolate human Leydig cell mesenchymal precursors. cell culture 988
human discontinuous (5-layer) testis Percoll gradient centrifugation permitted isolation of two Leydig cell fractions. cell culture 989
mouse continuous (linear) testis Two groups were obtained: group 1 had densities of 1.0667 to 1.0515 g/ml; group 2 had densities of 1.0514 to 1.0366 g/ml. in vitro testosterone production electron microscope stereology 990
porcine discontinuous testis Purity of Leydig cells was > 85%. effect of hydrocortisone (HS) and adrenocorticotropic hormone (ACTH) on testosterone production 991
rat continuous testis Rat Leydig cells were purified from testis using elutriation followed by Percoll gradient centrifugation.   cell culture, the effect of human chorionic gonadotropin (hCG) on its gene regulation and protein secretion 992
rat continuous testis   cell culture, the effect of GHreleasing hormone (GHRH) on Leydig cell steroidogensis 993
rat continuous testis Rat Leydig cells were purified from testis using elutriation followed by Percoll gradient centrifugation. Band 2(of 3) contained > 95% Leydig cells (average density was 1.075 g/ml).   cell culture in presence of 125Ilabeled hCG, testosterone and cAMP production 994
rat continuous testis Comparison of Leydig cells of different densities were made. viability staining, cell culture 995
rat continuous testis   viability staining, in vitro testosterone production, SDSPAGE electrophoresis 996
rat continuous testis Isolation by Percoll gradient resulted in complete retention of morphological and biological integrity and a purity of 90 to 95%. cell culture in presence of human chorionic gonadotropin (hCG), phase contrast microscopy, light microscopy and electron microscopy 31
rat discontinuous (2-step) testis   cell culture in the presence of interleukin-1 (IL-1) 997
rat continuous (selfgenerating) testis Leydig cell precursors and pure (96%) Leydig cells were isolated on Percoll gradients. cell culture in presence of human chorionic gonadotropin (hCG) 998
rat discontinuous testis The purity of Leydig cells ranged from 90 to 95%. cell culture in presence of human chorionic gonadotropin (hCG) 999
rat discontinuous and continuous testis In the discontinuous gradient, the densest fraction contained a high proportion of Leydig cells whereas the lighter fraction contained mostly non-Leydig cells. 125I-labeled iododeoxyuridine incorporation 1000

 

Spermatozoa
Species Gradient type Comments Downstream application Ref. #
bovine discontinuous Percoll was thought to improve semen and preserve acrosome integrity. acrosome microscopy evaluation 1024
hamster continuous Caput epididymal spermatoazoa, with a specific gravity of 1.10-1.12 g/ml, were isolated without contamination by other cells. lipid extraction and fractionation electron microscopy 1025
macaque continuous Percoll separation resulted in increased spermzona binding and did not affect the percentage of acrosome-reacted sperm bound to the zona or the percent motility and percentage of acrosome-reacted sperm in suspension. zona binding experiments, acrosome reaction, motility assays 1026

 

Bone marrow cells
Species Gradient type Tissue type Comments Downstream application Ref. #
normal human discontinuous (2-layer) bone marrow Megakaryocytes were at the interface between 1.020 g/ml and 1.050 g/ml. magnetic beads for further purification, flow cytometry 1027
normal human discontinuous blood B cells were recovered at least 95% pure. Gradients removed B-cell blasts very effectively. flow cytometry 1028
HIV infected, normal and immune throm-bocytopenic purpura human discontinuous (2-layer) bone marrow Cells at the 1.020/1.050 interface were enriched 10-fold in megakaryocytes, while those at the 1.050/1.070 interface were immature cells.   megakaryocyte cultures prepared from immature cells for in situ hybridization 1029
normal human discontinuous (2-layer) bone marrow Percoll density fractionation resulted in the depletion of greater than 95% of total marrow cells and an increase in megakaryocyte frequency from about 0.05% to 3 to 7%. preparation of RNA and subsequent PCR, flow cytometry 1030
normal and arthritic human discontinuous (3-layer) bone marrow Cells prepared were suitable for cell culture. colony plaque assay, immunoflourscence, flow cytometry, protein colony blotting, RNA-colony blotting 1031
normal and leukemic human discontinuous (4-layer) peripheral blood Low density cells post- and pre-transplant were prepared for analysis. magnetic beads for further purification, PCR 1032
normal human discontinuous (7-layer) bone marrow T cells obtained using Percoll were enriched about two-fold in the high- density fractions of marrow cells and depleted by about four- to five-fold in the lowest-density fraction as compared with Ficoll™. flow cytometry, mixed lymphocyte reaction assay, natural killer cell assay, cell culture 1033
normal human discontinuous (1-layer) bone marrow Bone marrow cells were prepared using Percoll to remove RBC.   isolation of CD34+ cells using soybean agglutinin-coated flasks, progenitor cell assays, and flow cytometry 1034
marmoset discontinuous (1-layer) bone marrow Bone marrow megakaryocytes from both interleukin-6 (IL-6) treated and untreated animals could be separated in Percoll. flow cytometry 1035
primate discontinuous (1-layer) bone marrow Bone Marrow was isolated from both normal monkeys and interleukin-6 (IL-6) treated monkeys. cell enumeration, FACS, digital imaging microscopy and electron microscopy 1036
monkey discontinuous (1-layer) bone marrow peripheral and blood Light density cells were prepared from aspirates over a 60% cushion. cell culture and identification of various colony types 1037
mouse discontinuous (1-layer) bone marrow Red blood cells were removed from bone-marrow preparations with a single 70% Percoll cushion. culture of hematopoietic precursers, effects of interleukin-10 (IL-10) on proliferation, alkaline phosphatase activity, collagen synthesis assay, osteocalcin, preparation of RNA, and electron microscopy 1038
mouse discontinuous (3-layer) bone marrow Bone marrow progenitor cells were suitable for culture. effects of interleukin-3 (IL-3) and lipoplysaccharide (LPS) on cultured cells 1039
mouse discontinuous (3-layer) bone marrow Cells prepared were depleted of lymphoid and macrophage-lineage cells by addition of monoclonal antibody plus complement. FACS analysis, hematopoietic progenitor cell culture, reconstitution of lethally irradiated mice 1040
mouse discontinuous (3-layer) bone marrow Percoll was used to separate bone marrow fractions containing mostly blasts and lymphoid cells from those containing a high level of colony-forming units-spleen (CFU-S) counts. FACS analysis, chemotaxis assay, assay of colony-forming units-spleen (CFU-S) 1041
mouse discontinuous (3-layer) proteasetreated calvarial bone sections Percoll gradients gave distinct subpopulations of cells based upon the results of various assays. primary cell culture, flow cytometry, insulin-like growth factor I (IGF-I) assay, binding of epidermal growth factor, alkaline phosphatase determination 1042
mouse discontinuous (4-layer) bone marrow Normal suppressor cell activity was maintained after separation.   suppressor cell activity assay 1043
mouse discontinuous (4-layer) bone marrow Cells at a 1.06/1.07 g/ml density were used in subsequent studies. reconstitution of lethally irradiated animals 1044
mouse discontinuous (5-layer) bone marrow, spleen   flow cytometry, reconstitution of lethally irradiated mice 1045
rat discontinuous (3-layer) bone marrow About 75% of the input CFUmegakaryocytes (CFU-MK) were recovered in the fraction between 1.063 and 1.082 g/ml Percoll. CFU-MK were enriched only in this density fraction. culture of hematopoietic progenitor cells 1046
rabbit continuous bone marrow   implantation into in vivo placed diffusion chamber, cytochemical staining, and electron microscopy   38
feline discontinuous (1-layer) bone marrow Marrow mononuclear cells from both feline immunodeficiency virus-infected cats and normal cats were isolated. culture of hematopoietic progenitor cells 1047

 

Macrophages
Species Gradient type Tissue type Comments Downstream application Ref. #
human discontinuous lung Alveolar macrophages were purified from contaminating granulocytes using a discontinuous Percoll gradient. superoxide (SO) release 1048
human discontinuous (4-layer) brochoalveolar lavage Percoll gradients gave > 95% alveolar macrophage (AM) purity. cell viability assay, light microscopy 1049
human discontinuous (4-layer) lung Use of Percoll resulted in near total purification of alveolar macro-phages (AM) from other cells. superoxide (SO) anion release 1050
human discontinuous (4-layer) decidual tissue When cells were purified further with Percoll, the percentage of CD-14- positive cells increased by 52%. secretion of plateletactivating factor (PAF) acetylhydrolase 1051
human discontinuous pulmonary > 97% of the cells of fractions 1 to 4 were (4-layer) shown to be alveolar macro-phages (AM) in a previous study. nonspecific esterase staining, flow cytometric DNA analysis 1052
human discontinuous (4-layer) lung This method was used to study alveolar macrophage (AM) heterogeneity. The increased numbers of hypodense AM found in the asthmatic patients were unlikely to be due to the procedure.   cell viability, esterase and peroxidase activity assays, electron microscopy, generation of superoxide anion and thromboxane B2 1053
human discontinuous (5-layer) peripheral blood Percoll-isolated monocyte/macrophages were harvested from the top layer and routinely contained 75/90% monocytes/macrophages as identified by Wright-Giemsa stain. interactions between monocyte/macrophage and vascular smooth muscle cells 928
mouse continuous and discontinuous peritoneum The total cell yield was 100.0% ±0.8%, and as measured by the trypan blue exlusion test, the cell viability was completely preserved.   light microscopy, trypan blue exclusion, esterase activity assay, peroxidase activity assay, cell immunophenotyping, bacterial phagocytic assays 1054
mouse discontinuous (4-layer) cultured cells Percoll did not have a detectable effect on the cytolytic activity of cultured macrophages or on their viability. phagocytic and cytolytic assays 30
mouse, rat continuous and discontinuous peritoneum A continuous gradient followed by a discontinuous gradient was used to isolate all cell populations according to their actual density. This procedure yielded cells of high viability with preservation of critical cell function trypan blue exclusion 1055
rat discontinuous (5-layer) lung The Percoll fractions were designated I to IV in order of increasing density with a percent distribution of cells of about 5, 15, 50 and 30%, respectively. Cell viability was > 95%.   fluorescence microscopy 1056
rat discontinuous (5-layer) lung Cell viability was > 95% by trypan blue exclusion and > 95% were identified as alveolar macrophages (AM) in un-fractionated and fractionated cells by Giemsa and nonspecific esterase stains. effects of pulmonary surfactant and protein A on phagocytosis, light microscopy 1057
rat continuous bronchoalveolar lavage The various fractions comprised approximately 90 to 99% macrophages in virtually all instances. esterase activity, surface expansion of Ia antigen by an immunoperoxidase technique 1058

 

Mast cells
Species Gradient type Tissue type Comments Downstream application Ref. #
mouse NA peritoneum Purity of the mast cells was nearly 100%, as checked by Memacolor fast staining. qualitative and quantitative PCR analysis 1059
mouse continuous peritoneum Starting from a peritoneal cell population containing 4% mast cells, a mast cell purification of up to 95% was obtained. electron microscopy and ultrastructural cytochemistry 8
rat discontinuous peritoneum Mast cell purity with Percoll was > 95%. direct interaction between mast and non-mast cells, histamine release assay 1060
rat continuous peritoneum Mast cells purified on Percoll gradients were more than 90% pure by toluidine blue staining, and the viability was > 98% by the trypan blue exclusion test. flourometric assay to measure histamine release 1061
rat continuous peritoneum Mast cells can be isolated with high yields and purity by centrifugation on gradients of Percoll. light and electron microscopy, cytofluorometry 9
rat continuous (sequential) peritoneum The purity of mast cells purified over sequential Percoll gradients was evaluated by measurement of the contribution of eosinophil peroxidase to mast cell peroxidase activity. histamine release and peroxidase activity 1062

 

Thymocytes
Species Gradient type Tissue type Comments Downstream application Ref. #
mouse discontinuous (5-layer) thymus Percoll was used for separation of immature thymocytes. in vitro stimulation by mitogens, isolation of nuclei, isolation and gel electrophoresis DNA, enzyme assays 1063
rat discontinuous thymus Percoll was used for separation of normal and apoptotic thymocytes. flow cytometry 1064
rat discontinuous (4-layer) thymus Percoll was used for separation of cells possessing the characteristically condensed nuclear chromatin associated with apoptosis from apparently normal thymocytes. electron microscopy, Coulter counter analysis, flow cytometry, DNA analysis 1065
rat discontinuous (4-layer) thymus Percoll was used for isolation of a transitional population of preapoptotic thymocytes. DNA analysis, isolation of nuclei and DNA autodigestion, light and electron microscopy 1066
rat, mouse discontinuous (3-layer) thymus Percoll was used to separate large and small thymocytes. An extremely high level of viability was maintained phase contrast microscopy and autoradiography 62

 

Miscellaneous cells
Cell Type Species Gradient type Tissue type Comments Downstream application Ref. #
pancreatic islets human, mouse continuous pancreas The use of Percoll eliminated the problems of high viscosity, undesired osmotic properties and, in some cases, also toxic effects. density determination and insulin secretion 5
endothelial human continuous linear gradient whole blood Final recovery of endothelial cells was 91.6%. immunofluorescence 1067
trophoblasts rat continuous placenta Percoll gradient centrifugation yielded efficient separation of rat placental lactogen- II (rPL-II) producing cells from digested tissue from labyrinth and junctional zones of the chorioallantoic placenta. development of in vitro rat placental trophoblast cell culture system 1068
various NA NA NA This paper compared different approaches to cell separation. According to the authors, Percoll is generally the most useful media for isopycnic centrifugation of most kinds of cells. none 1069
viable vs. nonviable human, rat discontinuous (2-layer) various tumor tissue Interface showed a viability of > 90%, but the yield of viable cells decreased dramatically if the tissue resection was not immediately processed. trypan blue viability assay, 2-D PAGE 1070
apoptotic human discontinuous (7-layer) promyelocytic leukemic cell line The step gradient used generated three main cell bands and a cell pellet, the pellet was very enriched for apoptotic cells (85 to 90%). DNA isolation 1071
lymphoblast human continuous whole blood Lymphoblasts were enucleated using a Percoll gradient containing cytochalasin B. electrofusion 1072
brain capillary endothelial rat continuous pre-made brain Subsequent Percoll gradient centrifugation resulted in a homogenous population of capillary endothelial cells capable of attachment to collagen and incorporation of tritiated thymidine. cell culture, light microscopy electron microscopy 170
neurons rabbit discontinuous and rate zonal dorsal-root ganglia Neurons were isolated with a viability of 80% and a purity of > 90%. cell culture, light and electron microscopy 480
nonmyogenic separated from myogenic chicken discontinuous breast muscles Separation of cells from embryonic muscle allowed direct analysis of cell-specific proteins without the need for cell culturing. cell culture, microscopy, DNA/ protein analysis 680
megakaryocytes human discontinuous bone marrow Isolation of megakaryocytes was reproducibly better in Percoll than in BSA. Ficoll 400 centrifugation to further purify, complement receptor assay 155
chondrocytes rat discontinuous bone marrow Cell viability was > 95% while yield varied depending on aggregation of cells. cell culture, quantitation of proteoglycans and collagen 629
spermiophages turkey discontinuous sperm Spermiophages fixed immediately after Percoll isolation resembled those in freshly ejaculated semen except for an apparent increase in the number of mitochondria. light and electron microscopy, cell culture 1073
NA human continuous parathyroid gland Densities of parathyroid glands were measured using various density gradient media. For densities > 1.0 g/ml, Percoll proved superior to any of the other gradient liquids investigated. glandular density determination 2

References can be found on pg 56-76 of the Cell Separation Media Methodology and Applications Handbook.

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