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HomeCell Counting & Health AnalysisApplications of Percoll In Blood Cells

Applications of Percoll In Blood Cells

Blood cells

The entire spectrum of cell types present in blood can be resolved on preformed gradients of Percoll. The method described by Pertoft et al. (55) (Fig 19) utilizes both rate zonal (separation by size) and isopycnic (separation by density) techniques. Diluted blood was layered on top of a preformed self-generated gradient and centrifuged for 5 min at 400 × g, during which time the thrombocytes or platelets (which are appreciably smaller than the other cells present) did not penetrate into the gradient.

The plasma layer containing the thrombocytes was removed and replaced by saline, and centrifugation was continued at 800 × g for 15 min, resulting in isopycnic banding of mononuclear cells (lymphocytes and monocytes), polymorphonuclear cells and erythrocytes. The position and densities of the banded cells were monitored using Density Marker Beads in an identical gradient contained in a second centrifuge tube.

Although the above method demonstrates the utility of Percoll for fractionating whole blood, most blood cells can be appreciably enriched using a simple step gradient. A simple step gradient often gives acceptable yields and purity for downstream processing. The Application Tables below contain a number of examples of purification of blood cells and other cell types using different types of Percoll gradients.

Separation of human blood cells in a gradient of Percoll

Figure 19.Separation of human blood cells in a gradient of Percoll. The tubes were filled with 10 ml of 70% (v/v) Percoll in 0.15 M NaCl (p=1.086 g/ml), and the gradient performed by spinning in a 14° angle rotor at 20 000 × g for 15 min. Two ml of gradient material was removed from the bottom of the tube using a syringe, and 1 ml of heparinized blood diluted with 1 ml of 0.15 M NaCl was layered on top of the gradient. Centrifugation was carried out as indicated. Densities were monitored using Density Marker Beads. MNC = Mononuclear cells, PMNC = Polymorphonuclear cells, RBC = Red blood cells (55, reproduced by kind permission of the authors and publisher).

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.

Lymphocytes
SpeciesGradient typeTissue typeCommentsDownstream applicationRef. #
humancontinuousbloodPercoll density centrifugation resulted in significant downregulation of L-selectin surface reactivity.Immunoflourescence891
humancontinuoustonsilA Percoll density gradient was used for separation of large (low density) in vivo activated cells from small (high density) resting cells.cell culture, FACS, granulocytemacrophage colony stimulating factor (GM-CSF) assays, and Northern blots892
humancontinuousspleen, tonsilLarge B lymphocytes from tonsils (in vivo activated cells) obtained by Percoll gradient centrifugation displayed higher IL-4R levels than resting cells.cell culture, Northern blots, FACS893
humancontinuousperipheral bloodPercoll was used to separate proliferating form nonproliferating cells.tritiated thymidine incorporation11
humancontinuoustonsil, peripheral bloodThis procedure yielded > 90% viable cells and has proved quite helpful in renewing overgrown cultures.proliferation and cytotoxicity assays16
humancontinuousbloodPercoll was used to separate monocytes from lymphocytes.cell culture, coagulation activity, immunoradiometric assays40
humandiscontinuous (3-layer)intestineLymphocytes were enriched in the interface between 66.7 and 44% Percoll. Further purification was performed using magnetic beads.flow cytometric analysis, immunoperoxidase procedure, cell culture894
humandiscontinuous (6-layer)peripheral bloodPercoll was used to separate large granular lymphocytes (LGL) from peripheral mononuclear cells.detection of CD5LOW+ in the LGL population895
humandiscontinuoustonsilPercoll gradient was used for the separation of small (high density) and large (low density) cells.cell culture, apoptosis assays, immunoassay for G-CSF, bioassay for GM-CSF, Northern blot analysis 896
humandiscontinuousintestine proliferation assays, measurement of cytotoxicity, H1 receptor binding studies897
humandiscontinuousperipheral bloodPercoll was used for the isolation of low density cells.FACS, immunoflourescence, nonspecific esterase staining898
humandiscontinuousperipheral bloodLymphoctes were recovered from low density Percoll fractions.suppression of NK-cell proliferation by freshly isolated monocytes899
humandiscontinuoustonsilPercoll was used to isolate follicular dendrite cells (FDCs).cell sorting, B cell proliferation by FDCs900
humandiscontinuousbone marrowPercoll was used to isolate leukemic cells from bone marrow.establishment of a leukemic cell line901
humandiscontinuousperipheral bloodAfter separation on Percoll, a virtually pure population of activated cells was obtained, as estimated by the presence of the 4F2 marker and of the transferrin receptor.immunoflourescence and assay of phospholipid metabolism902
humandiscontinuous (1-step)bloodLymphocyte purity was > 99% and the population of monocytes was enriched 82 to 90%.induction and assay of lymphokine (IL-2)-activated killer (LAK) cell activity903
humandiscontinuous (4-layer)bloodPercoll was used for separation of large granular lymphocytic (LGL) cells from T cells.Giemsa staining, cell activation with interleukin-2 (IL-2)904
humandiscontinuous (4-layer)tonsilPercoll was used for B cell enrichment.flow cytometry905
humandiscontinuous (5-layer)bloodLarge granular lymphocytes (LGL) were collected from the low density fractions, whereas T cells were located in the higher density bottom fraction.FACS, cell culture, cytotoxicity assays906
humandiscontinuous (5-layer)bloodMonocytes were purified up to 90% and lymphocytes to > 99%.cell counting (hemocytometer) and cell culture assays69
humandiscontinuous (7-layer)peripheral blood cytotoxicity assay, flow cytometry analysis, and complement-dependent lysis907
humanselfgeneratingperipheral bloodPercoll was used to separate viable and nonviable cells. Yields were slightly higher and erythrocyte contamination was slightly lower with Percoll than with Ficoll- Isopaque.cytotoxicity assays83
caninecontinuousbloodPercoll was used for enrichment and depletion of antibody-positive cells.reverse hemolytic plaque assay and cell-mediated lympholysis908
caninediscontinuous (4-step and 2-layer)whole bloodA final sedimentation of purified lymphocytes through a 45/50% Percoll gradient concentrated natural killer (NK) activity into a single band of lymphocytes.measurement of NK activity909
mousecontinuous (3-layer)intestineEnrichment increased from 44.1% (single filtration) to 52.4% (multiple filtration) after nylon wool filtration, and from 70.3% (single filtration) to 82.8% (multiple filtration) after Percoll fraction.flow cytometry910
mousecontinuous (5-layer)spleenPercoll was used for separation of virgin and memory T cells.cell proliferation assays, FACS911
mousediscontinuous (3-layer)spleenPercoll was used for separation of B cells.protein phosphorylation assay912
mousediscontinuous (3-layer)intestinePercoll was used for isolation of intestinal intraepithelial lymphocytes (IEL).DNA analysis by flow cytometry, mRNA-cDNA dot blots, PCR913
mousediscontinuous (4-layer)spleenPercoll was used for isolation of small, resting B cells.cell cycle analysis by flow cytometry914
bovinediscontinuousmammaryPurified cells were > 80% pure.Wright´s Giemsa staining, cell culture915
Monocytes
SpeciesGradient typeTissue typeCommentsDownstream applicationRef. #
humandiscontinuous (minigradient)peripheral blood With the Percoll minigradient, cells could be obtained in 90 to 100% from the patients at all time points after bone marrow transplant (BMT).cytogenic analysis916
humancontinuousblood The isolated mononuclear leukocyte (MNL) fraction contained > 80% cells giving a positive reaction for a-naphthyl acetate esterase ( a-NAE).cell culture917
humancontinuousperipheral bloodPercoll was used to isolate monocytes with > 85% purity and > 95% viability.cell culture with cytokines918
humancontinuousbloodPercoll has proved very practical for the separation of monocytes from blood and of macrophages from ascites and synovial fluids.cell culture34
humancontinuousbloodPercoll gradients were used for the separation of monocytes from lymphocytes.cell culture40
humancontinuousbloodA one-step procedure was used for obtaining a high-yield suspension of monocytes of 20% purity, which does not require washing before cultivation. A two-step method gave better than 90% pure monocytes at a lower yield.cell counts, Fc-receptor presence and phagocytosis assays57
humancontinuousperipheral bloodMNL were separated into two fractions with Percoll: one consisting mostly of monocytes and the other lymphocytes.fungal (Coccidioides immits) killing assay919
humandiscontinuousbloodMonocyte purity was 95%.cell culture920
humandiscontinuouswhole bloodPercoll gradient was used for enrichment of hematopoietic progenitor cells.assay for colony formation921
humandiscontinuousblood RNA isolation, Northern blot analysis and RT-PCR922
humandiscontinuousbone marrow DNA hybridization studies923
humandiscontinuous (1-layer)bloodLymphocyte purity was > 99% and the population of monocytes was enriched 82 to 90%.induction and assay of lymphokine (IL-2)-activated killer (LAK) activity903
humandiscontinuous (1-layer)bloodPMN recovery was > 90% and RBC contamination < 5%.Northern blot analysis924
humandiscontinuous (1-layer)peripheral bloodMonocytes were ≥ 95% pure.Northern blot analysis, nuclear runoff experiments, S1 protection assay925
humandiscontinuous (4-layer)peripheral bloodCells obtained from the 65% to 75% interface were 99% granulocytes.analysis and Western blot analysis genomic DNA isolation and PCR926
humandiscontinuous (1-layer)peripheral bloodWith the 1-step gradient, the purity of the monocytes was 93 to 96%.Giemsa staining and cell culture927
humandiscontinuous (5-layer)peripheral bloodPercoll-isolated monocyte/ macrophages as identified by Wright-Giemsa stain.interactions between monocyte/macrophage and vascular smooth muscle cells928
humandiscontinuous (5-layer)bloodMonocytes were purified up to 90% and lymphocytes to 99% purity.cell recovery counting and cell culture assays69
humandiscontinuousperipheral blood cell enumeration with Coulter counter, RNA isolation, and Northern blot analysis929
equinediscontinuous (1-layer)peripheral bloodAll MNCs were recovered on Percoll gradients without any neutrophil contamination.cell recovery assays930
Erythrocytes
SpeciesGradient typeTissue typeCommentsDownstream applicationRef. #
human continuouswhole bloodPercoll was used for separating young and old erythrocytes.immunoflourescence analysis of complement receptor type 1 (CR1) and CD59, proteolytic cleavage of CR1 in vivo.931
human continuousbloodPercoll was used to separate Plasmodium falciparum-parasitized erythrocytes from nonparasitized erythrocytes.isolation of erythrocyte membranes lipid peroxidation, vitamin E and transmembrane reducing system analysis932
human continuousbloodA rapid method for the age fractionation of human erythrocytes by Percoll density gradient centrifugation was described.flame photometry, enzyme assays77
human discontinuous (4-layer and 8-layer)bloodA rapid method using Percoll to fractionate erythrocytes according to age was described.analysis of the decline of enzymatic activity in aging erythrocytes933
human discontinuous (4-layer)blood ELISAs, proteolytic digestion of membranes934
human discontinuous (4-layer)bloodThe position of Density Marker Beads (Cytiva) was used to collect cells with densities < 1.00 g/cm3 or > 1.119 g/cm3.yield stress experiment: a sensitive index of cell: cell adhesion of deoxygenated suspensions of sickle cells935
human discontinuousbloodPercoll gradient was used to separate erythrocytes into 4 density fractions.platelet-activating factor (PAF) acetylhydrolase activity and membrane fluidity936
human discontinuousbloodErythrocytes loaded with L-asparaginase using a hypotonic dialysis process were separated into eight fractions.L-asparaginase activity937
human discontinuous bloodDiscontinuous gradient of the range 1.080 to 1.115 g/cm3 with each layer differing in density by 0.005 g/ml produced nine cell fractions.enzyme assays66
human discontinuous (5-layer)blood study of RBC deformability and cell age938
human discontinuous (8-layerbloodPercoll was used for density separation of RBC loaded with inositol hexaphosphate (IHP) by reverse osmotic lysis.haemoglobin distribution, distribution of IHP concentrations939
human discontinuous (9-layer)bloodA detailed comparison between two cell-loading techniques for inositol hexaphosphate was performed by monitoring the RBC distribution patterns on Percoll density gradients.oxygen affinity, hematological parameters and organic phosphate content measurements 940
Mastomys natalensiscontinuousbloodPercoll was used to separate Plasmodium berghei-paraitized erythrocytes from non parasitized cells.cAMP level in RBCs941
mousecontinuous (self-forming)bloodFractionation of RBC yielded five distinct populations that maintained their densities upon recentrifugation in a second gradient.transbilateral movement and equilibrium distribution of lipid 942
mousecontinuousperipheral bloodPercoll was used for density gradient separation of chemicallyinduced erythrocytes.fixing, staining and flow cytometric analysis of micronucleated polychromatic (MPCE) and micronucleated nonchromatic (MNCE) erythrocytes943
mousediscontinuousperipheral bloodErythrocytes were contaminated with only 0.001% nucleated cells.glucose phosphate isomerase (GPI) assay944
ratdiscontinuouswhole bloodPercoll was used to separate Plasmodium berghei-infected RBCs.oxygen dissociation analysis945
rabbitdiscontinuous (7-layer)bloodRabbit red blood cells were reproducibly fractionated into populations of various stages of maturation.measurement of cytosolic protease activities946
troutdiscontinuousbloodThe gradient in the region of 45 to 65% Percoll produced three red cell fractions which is due to multiplicity of haemoglobin components.antioxidant enzyme activities and membrane fluidity analysis947
Natural Killer (NK) cells
SpeciesGradient typeTissue typeCommentsDownstream applicationRef. #
humandiscontinuousperipheral bloodThe Percoll (preculture) step facilitated the density separation of resting cells from larger lymphocytes.NK- and T-cell activation, immunoflourescence948
humandiscontinuousbloodK562 cells which adhere to NK cells were separated together. Enrichment of NK cells was 71.3%.cytotoxicity studies, morphological characterization61
humandiscontinuous (2-layer)peripheral bloodThe low density fraction (42.5 to 47.5% Percoll) which showed a 4-fold enrichment in NK activity was used.NK activity and kinetic constant determinations, measurement of the effect of divalent cations on NK activity, and effect of ATP on NK cell-surface markers949
humandiscontinuous (6-layer)bloodFurther purification using magnetic beads resulted in a pure preparation.cytotoxic assay950
humandiscontinuous (8-layer)peripheral bloodRecovery was > 80% while viability, as judged by trypan blue exclusion, was > 95%.NK cell stimulatory effect, phenotype evalution by immunoflourescence951
mousediscontinuous (3-layer)lungThe cells at the 50/55% interface were the richest in NK cell activity.adoptive transfer to reconstitute NK activity in NKdepleted mice952
mousediscontinuous (6-layer)spleenNK cells were enriched in the lower density Percoll fraction, while natural cytotoxic T cells (NCT) were distributed between both higher and lower density fractions.cytotoxicity of NK cells was measured953
mousediscontinuous (6-layer)liverAll NK activity was above 1.08 g/ml density. Interfaces at 1.04 and 1.06 gave a 2× enrichment of NK progenitors.PCR, Western blot analysis, and cytotoxicity assays954
Neutrophils
SpeciesGradient typeTissue typeCommentsDownstream applicationRef. #
humandiscontinuous (1-layer)whole bloodNeutrophils pelleted in the 1.077 g/ml cushion.FACS analysis, intracellular Ca++ and superoxide anion measurements955
humandiscontinuous (2-layer)whole blood polymorphonuclear neutrophil (PMN) labeling by immunoflourescence, adherance assay and superoxide assay956
humandiscontinuous (4-layer)peripheral bloodPercoll was used to separate monocytes and lymphocytes.immunoflourescence and flow cytometry957
humandiscontinuous (4-layer)whole bloodEosinophils and neutrophils were isolated following dextran sedimentation.flow cytometry and measurement of lactoferrin release958
humandiscontinuousbloodCell preparation was layered onto a Percoll cushion to remove monocytes. After lysis of the erythrocytes, primarly neutrophils, with the remaining cells being predominantly eosinophils.immunoflourescence studies959
humandiscontinuousbloodThe neutrophils were > 95% pure.indirect immunoflourescence, immunoelectron microscopy and FACS analysis, O2 consumption960
humancontinuous, nonlinear (2-layer)bloodPercoll was used for subcellular fractionation of azurophil granules, specific granules, gelatinase granules, plasma membranes, and secretory vesicles.ELISAs for NGAL, gelatinase, lactoferrin and myeloperoxidase961
mousecontinuousperitoneumAn ~97% pure polymorphonuclear neutrophilic leukocyte (PMN) preparation was obtained using Percoll.electrophoretic analysis, GM-CSF assay, and cell morphology and counts70
Eosinophils
SpeciesGradient typeTissue typeCommentsDownstream applicationRef. #
humandiscontinuousperipheral bloodEosinophils were purified using Percoll gradients followed by immuno-magnetic beads. Using this procedure, the eosinophil purity was always > 95% and the viability was > 98%.FACS analysis, eosinophil migration assays, Ca++ measurements962
humandiscontinuous (2-layer)bloodThe recovery of eosinophils was 40 to 60%, the viability > 98% as tested by trypan blue exclusion, and the purity > 85%.chemotaxis and intracellular Ca++ measurements963
humandiscontinuous (2-layer)bloodEosinophil purity was > 95%, and the method did not induce priming of the eosinophils.serum-treated Zymosan (STZ) binding and placentaactivating factor (PAF) measurments964
humandiscontinuous (2-layer)bloodEosinophil purity was always > 85% and the recovery ranged from 40 to 60%. Viability was > 98%.chemotaxis assay965
humandiscontinuous (3-layer)peripheral bloodEosinophil purity was 95 to 99%, viability using trypan blue was > 98%, and recovery was 40 to 60%.density distribution analysis, cell culture966
humandiscontinuous (4-layer)whole bloodThe effect of dextran sedimentation on the density of neutrophils and eosinophils was analyzed.flow cytometry and measurement of lactroferrin release958
Basophils
SpeciesGradient typeTissue typeCommentsDownstream applicationRef. #
humancontinuousperipheral bloodBasophils were purified by Percoll density gradient separation and cell sorting. The procedure yielded 95% purity with a total yield estimated to range from 5 to 28%.flow cytometry, histamine release, electron microscopy967
humancontinuousbone marrowThe purity of basophils in the low density fraction (< 1.063 g/ml) was generally > 75% of the cells.histamine content and release968
humandiscontinuousperipheral bloodHighly purified basophils were obtained by Percoll gradient followed by negative selection using flow cytometry.effects of cytokines on human basophil chemotaxis969
humandiscontinuous (2-layer)bloodThe majority of the basophils were located at the 1.070 to 1.080 interface. The purity in this fraction was 36 to 63%.further purification by negative selection using immunomagnetic beads970
humandiscontinuous (2-layer)bloodHighly purified basophils were obtained by Percoll gradient followed by negative selection using flow cytometry.histamine release assay, chemotactic assay971
humandiscontinuous (3-layer)whole bloodBasophils were purified to > 80% using Percoll gradient followed by treatment with monoclonal antibodies to remove contaminants.flow cytometry and leukotriene C4 generation following calcium ionophore stimulation972
humandiscontinuous (3-layer)peripheral bloodBasophil purity was 85 to 96% using Percoll.cell stimuli and mediator release assay973
ratdiscontinuousblood further purification by immuno-magnetic beads, immunoflourescence, electron microscopy974
Materials
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