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Percoll gradient separation

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Percoll gradient separation is a laboratory technique used for the separation and purification of cells, organelles, and other biological particles. It utilizes a colloidal silica-based density gradient medium to achieve efficient and gentle separation based on differences in buoyant density. The core function of this product is to facilitate the isolation and enrichment of target cellular or subcellular components from complex biological samples.

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Lab products found in correlation

Gentamycin, by Thermo Fisher Scientific (2 mentions) Dnase 1, by Roche (2 mentions) Liberase, by Roche (2 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Collagenase d, by Roche (1 mentions) Human gm csf, by R&D Systems (1 mentions) Csf 1, by R&D Systems (1 mentions) Golgistop, by BD (1 mentions) Diva software, by BD (1 mentions) Aria 2, by BD (1 mentions) Lsrii cytometer, by BD (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Gm csf, by Miltenyi Biotec (1 mentions) Interleukin 4 (il 4), by Miltenyi Biotec (1 mentions) Interferon ifn γ, by R&D Systems (1 mentions) Lymphoprep, by Axis-Shield (1 mentions) Gm csf, by R&D Systems (1 mentions) Interleukin 4 (il 4), by R&D Systems (1 mentions) M csf, by R&D Systems (1 mentions)

Close spelling variants of this product

Percoll® density gradient separation medium Percoll gradient Percoll

8 protocols using percoll gradient separation

1

Tumor Dissociation and Cell Isolation

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The tumors were minced with surgical scissors and dissociated in an enzymatic solution of collagenase D (1 mg·mL−1) (Roche, Mannheim, Germany) and DNase I (100 μg·mL−1) for 30 min at 37 °C in a water bath with continuous agitation. After this treatment, the enzymatic solution was inactivated by adding DMEM with 10% FBS and was immediately centrifuged at 400 g for 5 min at 4 °C. The cell suspension was filtered through a 70‐μm cell strainer, subjected to discontinuous Percoll gradient separation (GE Healthcare, Pittsburgh, PA, USA). The cells were collected from their respective density fractions and prepared for flow cytometry analysis. One negative sample (no antibody) was used for gating purposes. Cell populations were determined by electronic gating based on forward versus side scatter. The CD45+ population was further characterized for T cells (CD3+/CD4+ and CD3+/CD8+) and neutrophils (CD11b+/Ly6G+). Flow cytometric data acquisition was performed using the LSRFortessa™ flow cytometer (BD Biosciences).
Xia Y., He J., Zhang H., Wang H., Tetz G., Maguire C.A., Wang Y., Onuma A., Genkin D., Tetz V., Stepanov A., Terekhov S., Ukrainskaya V., Huang H, & Tsung A. (2020). AAV‐mediated gene transfer of DNase I in the liver of mice with colorectal cancer reduces liver metastasis and restores local innate and adaptive immune response. Molecular Oncology, 14(11), 2920-2935.
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2

Macrophage Differentiation from Monocytes

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Human mononuclear cells were isolated from volunteer donor blood buffy coats (Sanquin), peripheral blood from healthy controls and RA patients, and from synovial fluid of RA patients by gradient centrifugation with Lymphoprep (Axis-Shield PoPAS) and monocytes were further isolated by Percoll gradient separation (GE Healthcare). Monocytes were differentiated into macrophages in IMDM/10 % fetal calf serum (FCS) supplemented with 100 μg/ml gentamycin (Invitrogen), in the presence of human GM-CSF (5 ng/ml, R&D Systems), CSF-1 (25 ng/ml, R&D Systems), or IL-34 (25 ng/ml, provided by Five Prime Therapeutics Inc.) for 7 days.
Garcia S., Hartkamp L.M., Malvar-Fernandez B., van Es I.E., Lin H., Wong J., Long L., Zanghi J.A., Rankin A.L., Masteller E.L., Wong B.R., Radstake T.R., Tak P.P, & Reedquist K.A. (2016). Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis. Arthritis Research & Therapy, 18, 75.
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3

Isolation and Characterization of Colonic and Splenic Immune Cells

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Colons were collected, flushed and enzymatically processed as previously described (2 (link)). Briefly, minced distal colons were washed 3 times for 20 min at 37oC in 2mM EDTA, 10% FCS, 25mM Hepes, HBSS buffer and subsequently digested using a Liberase/DNAse 1 (Sigma-Aldrich, St Louis, MO). Mononuclear cells were isolated by Percoll gradient separation (GE Healthcare Life Science, Pittsburgh, PA). Splenocytes were isolated from enzymatically dissociated spleen using Lymphoprep density gradient (Accurate Chemical & Scientific Corporation, Westbury, NY). For cytokine intracellular staining (ICS), cells were stimulated for 4.5 hours with phorbol 12-myristate 13-acetate (PMA) (30 nM), ionomycin (1 μM) in presence of monensin (GolgiStop, BD Biosciences, San Jose, CA). Antibodies used for staining are listed in Supplementary Table S1. A live/dead (L/D) dye (Life Technologies, Carlsbad, CA) was used to exclude dead cells. FACS data were acquired using a LSRII cytometer (BD Biosciences) and analyzed with DIVA software (BD Biosciences). In some experiments, populations were cell sorted using Aria II (BD Bioscience). To block IL-17, mice were injected intra-peritoneally with 150ug of anti-IL17a mAb (Rat IgG1κ, clone TC11-18H10.1, Biolegend, San Diego, CA) or isotype control mAb twice weekly as described in each figure.
Housseau F., Wu S., Wick E.C., Fan H., Wu X., Llosa N.J., Smith K.N., Tam A., Ganguly S., Wanyiri J.W., Iyadorai T., Malik A.A., Roslani A.C., Vadivelu J.S., Van Meerbeke S., Huso D.L., Pardoll D.M, & Sears C.L. (2016). Redundant innate and adaptive sources of IL-17 production drive colon tumorigenesis. Cancer research, 76(8), 2115-2124.
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4

Generation of Tolerogenic Monocyte-Derived Dendritic Cells

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Monocytes from LRSC were isolated by gradient centrifugation with Ficoll-Paque Plus (GE Healthcare) and, subsequently, Percoll gradient separation (GE Healthcare). After 2 h at 37°C, non-adherent cells were removed, and complete medium containing 1,000 U/ml GM-CSF and 800 U/ml IL-4 (Miltenyi) was added. One micromole of Dexamethasone (Dex) (Sigma) was added at day 3 or day 6 of differentiation to generate tolerogenic DC as described by Cabezón et al. (5 (link)). moDC phenotype at the end of differentiation was validated by flow cytometry monitoring of CD1a, CD14, and CD11c markers (Supplementary Figure 1).
Giroud P., Renaudineau S., Gudefin L., Calcei A., Menguy T., Rozan C., Mizrahi J., Caux C., Duong V, & Valladeau-Guilemond J. (2020). Expression of TAM-R in Human Immune Cells and Unique Regulatory Function of MerTK in IL-10 Production by Tolerogenic DC. Frontiers in Immunology, 11, 564133.
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5

Isolation of Mucosal and Immune Cells

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Small intestine and distal colon (Fig.S1A) were cut, washed and enzymatically digested (400 U/ml Liberase and 0.1 mg/ml DNAse1; Roche Diagnostics, Indianapolis, IN). Colon and blood leukocytes were isolated using Percoll gradient separation (GE Healthcare Life Science, Pittsburgh, PA). Splenocytes were isolated from Liberase-treated spleen samples using Lymphoprep density gradient (Accurate Chemical & Scientific Corporation, Westbury, NY).
Orberg E.T., Fan H., Tam A.J., Dejea C.M., Destefano-Shields C.E., Wu S., Chung L., Finard B.B., Wu X., Fathi P., Ganguly S., Fu J., Pardoll D.M., Sears C.L, & Housseau F. (2016). The Myeloid Immune Signature of Enterotoxigenic Bacteroides Fragilis-Induced Murine Colon Tumorigenesis. Mucosal immunology, 10(2), 421-433.
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6

Isolation of Mucosal and Immune Cells

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Small intestine and distal colon (Fig.S1A) were cut, washed and enzymatically digested (400 U/ml Liberase and 0.1 mg/ml DNAse1; Roche Diagnostics, Indianapolis, IN). Colon and blood leukocytes were isolated using Percoll gradient separation (GE Healthcare Life Science, Pittsburgh, PA). Splenocytes were isolated from Liberase-treated spleen samples using Lymphoprep density gradient (Accurate Chemical & Scientific Corporation, Westbury, NY).
Orberg E.T., Fan H., Tam A.J., Dejea C.M., Destefano-Shields C.E., Wu S., Chung L., Finard B.B., Wu X., Fathi P., Ganguly S., Fu J., Pardoll D.M., Sears C.L, & Housseau F. (2016). The Myeloid Immune Signature of Enterotoxigenic Bacteroides Fragilis-Induced Murine Colon Tumorigenesis. Mucosal immunology, 10(2), 421-433.
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7

Monocyte-Derived Macrophage Differentiation

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Human PBMCs were isolated from volunteer donor blood buffy coats (Sanquin) by gradient centrifugation with Lymphoprep (Axis-Shield PoPAS) and monocytes were further isolated by Percoll gradient separation (GE Healthcare). Monocytes were differentiated into macrophages in Iscove's modified Dulbecco's medium/10% fetal calf serum supplemented with 100 µg/ml gentamycin (Invitrogen), in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF, 5 ng/ml), macrophage colony-stimulating factor (M-CSF, 25 ng/ml), IL-4 (10 ng/ml), interferon (IFN)-γ (10 ng/ml) or IL-10 (10 ng/ml) (all from R&D Systems) for 7 d.
García S., Krausz S., Ambarus C.A., Fernández B.M., Hartkamp L.M., van Es I.E., Hamann J., Baeten D.L., Tak P.P, & Reedquist K.A. (2014). Tie2 Signaling Cooperates with TNF to Promote the Pro-Inflammatory Activation of Human Macrophages Independently of Macrophage Functional Phenotype. PLoS ONE, 9(1), e82088.
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8

Isolation of Mouse Immune Cells

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Cells were isolated from mouse spleen and cervical lymph node by mashing tissues between two frosted microscope slides. The cells were further treated with RBC lysis buffer (Gibco, catalog number: A1049201) to eliminate erythrocytes, washed, and resuspended in RPMI 1640 (Gibco, catalog number: 31800022) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 10 mM HEPES (Life Technologies, Waltham, MA, USA). Isolation of CNS mononuclear cells was achieved using Percoll-gradient separation (GE Healthcare Bio-Sciences, Uppsala, Sweden) as previous described (20 (link)).
Hu C.F., Wu S.P., Lin G.J., Shieh C.C., Hsu C.S., Chen J.W., Chen S.H., Hong J.S, & Chen S.J. (2021). Microglial Nox2 Plays a Key Role in the Pathogenesis of Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology, 12, 638381.
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