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Percoll density gradient centrifugation

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Percoll density gradient centrifugation is a laboratory technique used to separate and purify cells, organelles, or other biological particles based on their density. It involves the use of a colloidal silica-based medium that creates a continuous density gradient during centrifugation, allowing the separation of different components based on their specific density.

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Lab products found in correlation

Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Na ve cd4 t cell isolation kit 2, by Miltenyi Biotec (2 mentions) Edta vacutainer tube, by BD (2 mentions) Ficoll histopaque 1077 density gradient centrifugation, by Merck Group (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Collagenase 4, by Thermo Fisher Scientific (1 mentions) Mv bullet kit, by Lonza (1 mentions) Facsaria, by BD (1 mentions) Facsdiva, by BD (1 mentions) Dnase 1, by Merck Group (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) 25 cm2 cell culture flask, by Corning (1 mentions) Collagenase 4, by Merck Group (1 mentions)

Close spelling variants of this product

Percoll (850 citations) Percoll gradient (157 citations) Percoll density gradient (14 citations) Percoll gradient centrifugation (10 citations) Density gradient centrifugation (3 citations) Percoll density centrifugation (2 citations)

12 protocols using percoll density gradient centrifugation

1

Isolation and culture of primary cells

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Neonatal rat cardiomyocytes and CFBs as well as adult rat hepatocytes and aortic smooth muscle cells were isolated as previously described42 (link),43 (link), with minor modifications. Cardiac cells were isolated from 2 day-old Wistar rats, and cardiomyocytes and CFBs were purified by Percoll density gradient centrifugation (Sigma-Aldrich). These cells and J774.1 mouse peritoneal MQs (JCRB0018; JCRB Cell Bank) were cultured in DMEM with 10% FBS, antibiotics, and non-essential amino acids.
For bone marrow-derived MQs, bone marrow cells were isolated from 12 week-old male WT mice as previously described44 (link). The cells were then plated into 12-well plates at 1.5 × 106 cells/well in 1 mL of bone marrow differentiation media (RPMI1640 (Gibco) supplemented with 10% FBS, 10% L929-conditioned media, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine). On day 3, extra 1 mL of differentiation media were added to the cells and incubated for another 3 days. Human coronary artery endothelial cells were cultured as previously described4 (link). We used neonatal rat cardiomyocytes and adult rat hepatocytes without passage, and neonatal rat CFBs, adult rat aortic smooth muscle cells, and human coronary artery endothelial cells at passage 3 to 4 for experiments.
Hakuno D., Kimura M., Ito S., Satoh J., Nakashima Y., Horie T., Kuwabara Y., Nishiga M., Ide Y., Baba O., Nishi H., Nakao T., Nishino T., Nakazeki F., Koyama S., Hanada R., Randolph R.R., Endo J., Kimura T, & Ono K. (2018). Hepatokine α1-Microglobulin Signaling Exacerbates Inflammation and Disturbs Fibrotic Repair in Mouse Myocardial Infarction. Scientific Reports, 8, 16749.
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2

Isolation and Cryopreservation of Naive CD4+ T Cells

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Peripheral blood lymphocytes were isolated using Percoll density gradient centrifugation (Sigma-Aldrich) as previously described [8 (link)]. Naïve CD4+CD45RA+ cells were isolated by negative selection using the naïve CD4+ T-Cell isolation Kit II (Miltenyi Biotech, Bergisch-Gladbach, Germany). Briefly, total CD4+ T-cell population was depleted of non-Th cells and memory CD4+ T cells via magnetic beads loaded with biotin-conjugated monoclonal antibodies. Afterwards, T cells were frozen in IMDM containing 50% (v/v) FCS (Invitrogen) and 10% (v/v) dimethyl sulfoxide (Merck KGaA, Germany) for 7 days until use (survival rate after thawing: 60–80%).
Ashjaei K., Bublin M., Smole U., Lengger N., Hafner C., Breiteneder H., Wagner S, & Hoffmann-Sommergruber K. (2015). Differential T-Helper Cell Polarization after Allergen-Specific Stimulation of Autologous Dendritic Cells in Polysensitized Allergic Patients. International archives of allergy and immunology, 166(2), 97-106.
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3

Isolation and Culture of Murine Hepatic Stellate Cells

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HSCs were isolated from murine livers as previously described.11 (link) Briefly, the livers were perfused through the portal vein with collagenase IV (Life Technologies, Grand Island, NY, USA). The smashed cells were filtered through a nylon mesh. HSCs were purified by Percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA) and cultured in complete medium supplemented with 20% FBS (Gibco, Gaithersburg, MD, USA) for 7 to 14 days, unless otherwise indicated. The purity of HSCs ranged from 90% to 95%, as measured by desmin immunostaining and typical appearance of lipid droplets under a light microscope.
Huang W.H., Zhou M.W., Zhu Y.F., Xiang J.B., Li Z.Y., Wang Z.H., Zhou Y.M., Yang Y., Chen Z.Y, & Gu X.D. (2019). The Role Of Hepatic Stellate Cells In Promoting Liver Metastasis Of Colorectal Carcinoma. OncoTargets and therapy, 12, 7573-7580.
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4

Isolation and Characterization of Murine EPCs

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EPCs were isolated from 8-week-old male C57 mice. In brief, whole BM cells were collected under sterile conditions from femur (and tibias) by flushing the shaft with phosphate-buffered saline (PBS). The mononuclear cells were isolated by percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA) at 2,000 g for 20 minutes. After three rinses, the isolated cells were cultured in endothelial basal medium-2 (EBM-2) with an MV Bullet Kit (Lonza, Walkersville, MD, USA). After 7 days of culture, the EPC markers DiI-labeled acetylated low-density lipoprotein (DiI-AcLDL) (Sigma-Aldrich) and bandeiraea simplicifolia lectin 1 (BS-1 lectin) (Sigma-Aldrich) were confirmed by immunofluorescence analysis.
Zhang Z., Dong J., Lobe C.G., Gong P., Liu J, & Liao L. (2015). CCR5 facilitates endothelial progenitor cell recruitment and promotes the stabilization of atherosclerotic plaques in ApoE−/− mice. Stem Cell Research & Therapy, 6(1), 36.
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5

Isolation and Analysis of Immune Cells from Infected Mice

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Spleen and Liver were aseptically collected from 24 h infected mice. Splenocytes were obtained and labeled to distinguish DCs (CD11b+/CD11chigh) and macrophages (CD11b+/Ly6G−/Ly6C+/F4-80+), and sorted using a FACSAria and the FACSDiva software (BD Biosciences, NJ, USA) for posterior RNA analysis. Sorted cells purity was >95%.
Enriched Kupffer cells were obtained after hepatic collagenase digestion, followed by a Percoll density gradient centrifugation (Sigma-Aldrich, MO, USA) and a brief adhesion step, as described elsewhere (14 (link)). The percentage of Kupffer cells was determined by flow cytometry (F4/80+/CD11bvariable). Cells were resuspended in lysis buffer and frozen for posterior RNA analysis.
Pérez-Cabezas B., Cecílio P., Robalo A.L., Silvestre R., Carrillo E., Moreno J., San Martín J.V., Vasconcellos R, & Cordeiro-da-Silva A. (2016). Interleukin-27 Early Impacts Leishmania infantum Infection in Mice and Correlates with Active Visceral Disease in Humans. Frontiers in Immunology, 7, 478.
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6

Isolation of First Trimester Cytotrophoblasts

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Isolation of villous cytotrophoblasts from first trimester placenta was performed according to Haider et al. [29 (link)] and Vondra et al. [30 (link)] using three consecutive digestion steps followed by Percoll density gradient centrifugation (5–70%; Sigma-Aldrich, Taufkirchen, Germany). Placental tissues were washed with PBS and placental villi were minced. The digestion steps were performed in 50 mL tubes at 37 °C in a shaker with 200 rpm for 8 min, 15 min, and 15 min. The digestion buffer (10× HBSS (Hank’s Balanced Salt) [31 (link)], 7.5% NaHCO₃, 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) was supplemented with 0.25% trypsin (Gibco, Life Technologies, Carlsbad, CA, USA) and 1.25 mg/mL DNase I (Sigma-Aldrich, Taufkirchen, Germany). The second and third digestion steps were pooled and purified with a Percoll gradient as detailed for term placental cytotrophoblasts isolation (see below).
Kreis N.N., Friemel A., Jennewein L., Hoock S.C., Hentrich A.E., Nowak T., Louwen F, & Yuan J. (2021). Functional Analysis of p21Cip1/CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia. Cells, 10(9), 2214.
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7

PBMC Isolation from Whole Blood

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Whole blood (28 ml/cat) was collected by jugular venipuncture into EDTA Vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ). PBMC were isolated by Percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO) as previously described [42 (link)] or by Ficoll-Histopaque-1077 density gradient centrifugation (Sigma-Aldrich, St-Louis, MO) following the manufacturer’s guidelines. Single-cell suspensions were prepared from popliteal or submandibular peripheral lymph nodes (PLN) obtained through surgical biopsies by gently and repeatedly injecting sterile PBS into the tissue using an 18G needle until the cells were released from the tissue. Cell counts and viability were determined by trypan blue dye exclusion and viability was always >90%.
Miller M.M., Petty C.S., Tompkins M.B, & Fogle J.E. (2014). CD4+CD25+ T regulatory cells activated during feline immunodeficiency virus infection convert T helper cells into functional suppressors through a membrane-bound TGFβ / GARP-mediated mechanism. Virology Journal, 11, 7.
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8

Isolation and Expansion of Human Mesenchymal Stem Cells

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hMSCs were obtained according to a previously described method [24 (link)]. Briefly, approximately 8 ml bone marrow was aspirated from seven male and five female donors, 31–56 years old. Mononuclear cells were isolated by Percoll density gradient centrifugation (1.073 g/ml; Sigma, St. Louis, Missouri USA) at 900 × g for 20 minutes. The cells were rinsed with phosphate-buffered saline (PBS) and plated in a 25 cm2 cell culture flask (Costar Corning, NY, USA). The expansion medium used was Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; HyClone, Logan, Utah, USA) supplemented with 10 % fetal calf serum (HyClone) and 100 U/ml penicillin–streptomycin (HyClone). During culture, the medium was changed every 3 days. Cells were passaged after reaching 90 % confluence and then used for experiments (passage 1).
Wang H., He X.Q., Jin T., Li Y., Fan X.Y., Wang Y, & Xu Y.Q. (2016). Wnt11 plays an important role in the osteogenesis of human mesenchymal stem cells in a PHA/FN/ALG composite scaffold: possible treatment for infected bone defect. Stem Cell Research & Therapy, 7, 18.
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9

Isolation of Peripheral Blood Mononuclear Cells

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Whole blood (28 ml/cat) was collected by jugular venipuncture into EDTA Vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ). PBMC were isolated by Percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO) as previously described (32 (link)) or by Ficoll-Histopaque-1077 density gradient centrifugation (Sigma-Aldrich, St-Louis, MO) following the manufacturer’s guidelines. Single-cell suspensions were prepared from popliteal or submandibular peripheral lymph nodes (PLN) obtained through surgical biopsies by gently and repeatedly injecting sterile PBS into the tissue using an 18G needle until the cells were released from the tissue. Cell counts and viability were determined by trypan blue dye exclusion and viability was always >90%.
Miller M.M., Akaronu N., Thompson E.M., Hood S.F, & Fogle J.E. (2014). Modulating DNA methylation in activated CD8+ T cells inhibits Treg cell-induced binding of FoxP3 to the CD8+ T cell IL2 promoter. Journal of immunology (Baltimore, Md. : 1950), 194(3), 990-998.
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10

Isolation and Culture of Mouse Hepatic Stellate Cells

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HSCs were isolated from the mouse liver and prepared as previously described (9 (link),14 (link)). Briefly, the liver was perfused with collagenase IV (Sigma-Aldrich, St. Louis, MO) solution (1.0 mg/mL) via the portal vein. After 30 minutes of digestion at 37°C, the isolated cells were filtered through a nylon mesh (200 microns mesh size). The HSCs were enriched by Percoll density gradient centrifugation (Sigma-Aldrich; 1.130 relative density for 10 min at 39,000g) and cultured in RPMI 1640 complete medium supplemented with 20% fetal bovine serum for 14 to 21 days. The purity of HSCs exceeded 90% determined by desmin immunostaining.
Arakawa Y., Qin J., Chou H.S., Bhatt S., Wang L., Stuehr D., Ghosh A., Fung J.J., Lu L, & Qian S. (2014). Co-transplantation with Myeloid-Derived Suppressor Cells Protects Cell Transplants: A Crucial Role of Inducible Nitric Oxide Synthase. Transplantation, 97(7), 740-747.
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