Applications of Percoll in Other Cell Types

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