Discontinuous percoll

Manufactured by GE Healthcare

Sourced in United States

Discontinuous Percoll is a laboratory centrifugation medium used for the isolation and purification of cells, cell organelles, and other biological particles. It is a colloidal silica-based solution that forms a density gradient when centrifuged, allowing the separation of different cell types or subcellular components based on their density.

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6 protocols using discontinuous percoll

Enrichment of Microglia and Astrocytes

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At 14 h after the last cycle of RSD, microglia and astrocytes were isolated from whole brain homogenates as previously reported (Norden et al., 2014 (link)). In brief, brains were homogenized in phosphate-buffered saline (PBS, pH 7.4) by passing through a 70 μm cell strainer. The resulting homogenates were centrifuged at 900 × g for 6 min. Supernatants were removed and cell pellets were re-suspended in 70% isotonic Percoll (GE-Healthcare). A discontinuous Percoll (GE-Healthcare) density gradient (50%, 35%, and 0%) was overlaid and centrifuged at 2000 × g for 20 min. Enriched microglia were collected from the interphase between the 70% and 50% Percoll layers. These cells were characterized as enriched brain CD11b⁺ cells, as previous studies have demonstrated that viable cells isolated by Percoll density gradient yields >90% CD11b⁺ cells (Wohleb et al., 2011 (link), Wohleb et al., 2013 (link)). Enriched astrocytes were collected from the interphase between the 50% and 35% Percoll layers. As previously reported (Norden et al., 2014 (link)), 65–70% of the cells collected from this interphase were characterized as GLAST-1⁺ astrocytes.

Sawicki C.M., McKim D.B., Wohleb E.S., Jarrett B.L., Reader B.F., Norden D.M., Godbout J.P, & Sheridan J.F. (2014). Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain. Neuroscience, 302, 151-164.

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Isolation of Primary Mouse Hepatocytes

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Primary hepatocytes were isolated from mice as described previously [9 (link)]. Briefly, after being anesthetized with sodium pentobarbital, mice were perfused through the portal vein using Hanks’ Balanced Salt Solution without Ca²⁺ and Mg²⁺. Afterward, the liver was perfused with 0.05% collagenase type IV (Thermo Fisher Scientific, Waltham, MA, USA). The liver was excised and filtered through a 70 μm cell filter (BD Biosciences). Dissociated hepatocytes and non-parenchymal cells (NPCs) were separated using discontinuous Percoll (GE Healthcare, Piscataway, NJ, USA) gradient centrifugation. The cells were collected in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco) and cultured in an incubator at 37 °C under 5% CO₂.

Guo J., Song Z., Yu J., Li C., Jin C., Duan W., Liu X., Liu Y., Huang S., Tuo Y., Pei F., Jian Z., Zhou P., Zheng S., Zou Z., Zhang F., Gong Q, & Liang S. (2022). Hepatocyte-specific TMEM16A deficiency alleviates hepatic ischemia/reperfusion injury via suppressing GPX4-mediated ferroptosis. Cell Death & Disease, 13(12), 1072.

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Synaptosomes Conjugation and Astrocyte Uptake

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Synaptosomes were purified and conjugated as previously described [7 (link)–9 ]. Briefly, adult C57BL/6J mouse brain was dissected and the cortex isolated. Synaptosomes were purified from cortex homogenates using a discontinuous Percoll (GE Healthcare Life Sciences, Pittsburgh, PA, USA) gradient. Purified synaptosomes were further incubated with pHrodo red, succinimydyl ester (Life Technologies), in 0.1 M sodium carbonate (pH 9.0) at RT for 2 hours. After washing to remove unconjugated pHrodo, pHrodo-conjugated synaptosomes were re-suspended in Am-a medium. Astrocytes were incubated with pHrodo-conjugated synaptosomes for 24 hours. For flow cytometry analysis, cells were collected by trysinization and centrifugation, and re-suspended in Dulbecco’s PBS (Life technologies) containing 0.02% fetal bovine serum (FBS, Atlas Biologicals, Fort Collins, CO, USA). Live cells were analyzed using FC500 Flow Cytometer (Beckman Coulter Life Sciences, Indianapolis IN, USA) on the basis of pHrodo red fluorescence intensity.

Liu Y., Patel G.C., Mao W, & Clark A.F. (2018). Establishment of a conditionally immortalized mouse optic nerve astrocyte line. Experimental eye research, 176, 188-195.

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Murine Immune Cell Isolation and Stimulation

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Murine peritoneal cells were plated in DMEM/F-12 with GlutaMAX™ (Gibco™) and supplemented with 10% FCS; adherent cells were cultured for 24 h prior to stimulation with 10 ng/ml IFNβ (Pestka Biomedical Laboratories), 10 ng/ml IFNγ (Peprotech) or both for 18 h. CNS tissue obtained from perfused mice was digested in 1X HBSS (Sigma-Aldrich) supplemented with 0.05% collagenase D (Roche), 0.1 μg/ml TLCK (Sigma-Aldrich) and 0.025 U/ml DNase I (ThermoFisher Scientific). Mononuclear cells were then isolated by discontinuous Percoll (GE Healthcare Life Sciences) gradient centrifugation. Spleens and inguinal lymph nodes were obtained by standard methods. For culturing cells were plated in RPMI supplemented with 10% FCS, 2 mM glutamine, penicillin/ streptomycin and with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 0.5 μM beta-mercaptoethanol (GIBCO). For phosflow splenocytes were stimulated as for human PBMCs but with murine IFNβ (Pestka Biomedical Laboratories), IL-6, IL-10, IL-12 (Peprotech) or IL-23 (R&D Systems). For recall assays cells were stimulated for 72 h with 10 μg/ml MOG_35-55 and restimulated with PMA and ionomycin as for CD4⁺ T-cell differentiation.

Dendrou C.A., Cortes A., Shipman L., Evans H.G., Attfield K.E., Jostins L., Barber T., Kaur G., Kuttikkatte S.B., Leach O.A., Desel C., Faergeman S.L., Cheeseman J., Neville M.J., Sawcer S., Compston A., Johnson A.R., Everett C., Bell J.I., Karpe F., Ultsch M., Eigenbrot C., McVean G, & Fugger L. (2016). Resolving TYK2 locus genotype-to-phenotype differences in autoimmunity. Science translational medicine, 8(363), 363ra149.

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Enriching Immunocytes from Mouse Tumors

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After careful excision of mouse tumor tissues, portions were minced into 2-to 4-mm pieces. These tissues were further dissociated into single-cell suspensions by combining mechanical dissociation with enzymatic degradation of the extracellular matrix using a Mouse Tumor Dissociation Kit (Miltenyi Biotec Inc, Auburn, CA, USA). The discontinuous Percoll (GE Life Sciences, Pittsburgh, PA, USA) gradient (44% and 67%) separation method was used to enrich immunocytes.

Kim S.H., Jason R., Cho S., Ogata D., Milton D.R., Peng W., Menter D.G., Ekmekcioglu S, & Grimm E.A. (2018). The COX2 effector microsomal PGE2 synthase-1 is a regulator of immunosuppression in cutaneous melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(5), 1650-1663.

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Neutrophil Isolation from Peripheral Blood

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Peripheral venous blood was collected into EDTA tubes, layered on Histopaque 1119 (Sigma-Aldrich) and centrifuged for 20 min at 800x g. The plasma, PBMC and neutrophil layers were collected and neutrophils were washed in Hyclone Hank's Balanced Salt Solution (HBSS) without calcium, magnesium or phenol red (GE Healthcare) supplemented with 10mM HEPES (Invitrogen) 0.1% plasma and further fractionated on a discontinuous Percoll (GE Healthcare) gradient consisting of layers with densities of 1105 g/ml (85%), 1100 g/ml (80%), 1093 g/ml (75%), 1087 g/ml (70%), and 1081 g/ml (65%) by centrifugation for 20 min at 800x g. Neutrophil enriched layers were collected and washed.

Ιωάννου Μ., Hoving D., Aramburu I.V., Vasconcelos N.M.d., Temkin M.I., Wang Q., Vernardis S.I., Demichev V., Tsourouktsoglou T., Boeing S., Goldstone R., David S., Stahl K., Bode C., Ralser M., & Papayannopoulos V. (2021). SIGNR1 promotes immune dysfunction in systemic candidiasis by modulating neutrophil lifespan via T cell-derived histones and G-CSF.

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