Percoll density gradient

Manufactured by Merck Group

Sourced in United States

Percoll is a colloidal silica-based density gradient medium used for the separation and isolation of cells, organelles, and biological macromolecules. It is designed to provide a versatile and gentle method for density-based separations, without the need for organic solvents or extreme centrifugation conditions.

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Lab products found in correlation

Collagenase 4, by Roche (3 mentions) Collagenase d, by Roche (2 mentions) Dnase, by Merck Group (2 mentions) Fitc conjugated anti cd3, by Thermo Fisher Scientific (1 mentions) Pe conjugated anti cd4, by Thermo Fisher Scientific (1 mentions) Apc conjugated anti cd8 mabs, by Thermo Fisher Scientific (1 mentions) Tnf α, by Merck Group (1 mentions) Complete freund s adjuvant cfa, by Chondrex (1 mentions) Rat tail collagen, by Merck Group (1 mentions) Gentamicin, by Thermo Fisher Scientific (1 mentions) Glutamine, by Merck Group (1 mentions) Glutamine, by Thermo Fisher Scientific (1 mentions) Hepes ph 7, by Thermo Fisher Scientific (1 mentions) Epidermal growth factor (egf), by BD (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Collagenase 4, by Merck Group (1 mentions) Insulin, by Merck Group (1 mentions) Ethanolamine, by Merck Group (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Linoleic acid, by Merck Group (1 mentions)

Close spelling variants of this product

Percoll (850 citations) Percoll gradient (157 citations) Percoll density gradient centrifugation (12 citations) Percoll density gradient media (4 citations)

14 protocols using percoll density gradient

Isolation of Lamina Propria Lymphocytes

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For the isolation of LPMC from colons, Peyer’s patches were removed, lamina propria lymphocytes were isolated via incubation with 5 mM EDTA at 37°C for 30 min, followed by further digestion with collagenase IV and DNase at 37°C for 1 h. Cells were then separated with a Percoll density gradient (Sigma-Aldrich, St. Louis, MO, USA). Mesenteric LN and spleens were smashed into 70-µm nylon strainers (BD) and eritrocytes lysed with RBC Lysis buffer (BD). Livers were mechanically dissected and mononuclear cells separated with a Percoll density gradient (Sigma-Aldrich, St. Louis, MO, USA).
In some experiments, after isolation, cells were re-stimulated in vitro for 3 h with PMA/ionomycin in the presence of Brefeldin A to evaluate cytokine secretion.

Burrello C., Garavaglia F., Cribiù F.M., Ercoli G., Bosari S., Caprioli F, & Facciotti F. (2018). Short-term Oral Antibiotics Treatment Promotes Inflammatory Activation of Colonic Invariant Natural Killer T and Conventional CD4+ T Cells. Frontiers in Medicine, 5, 21.

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Synthesis and Characterization of PLGA-b-PEG-b-PLGA Copolymer

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PLGA-b-PEG-b-PLGA triblock copolymer (number-average molecular weight (M_{n, NMR}) = 4200 g/mol, LA/GA = 75:25, mol/mol) was synthesized as our previous works [30 (link)]. Percoll density gradient, 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and anti-collagen type II antibody (anti-COL-II Ab) enzyme-linked immunosorbent assays (ELISAs) kit were purchased from Sigma-Aldrich, USA. Chick type II collagen (COL-II) and complete Freund's adjuvant (CFA) were acquired from Chondrex, USA. FITC-conjugated anti-CD3, PE-conjugated anti-CD4, and APC-conjugated anti-CD8 mAbs were obtained from eBioscience, San Diego, CA. Dimethyl sulfoxide (DMSO) was stored over calcium hydride (CaH₂) and purified by vacuum distillation with CaH₂.

Liu H., Ding J., Wang J., Wang Y., Yang M., Zhang Y., Chang F, & Chen X. (2015). Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells. PLoS ONE, 10(3), e0120596.

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Isolation and Culture of Primary Mouse Hepatocytes

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Primary mouse hepatocytes (PMoH) were isolated from up to 12-week-old C57BL/6 mice, as previously described[12 (link)]. In brief, PMoH were extracted via ex-vivo perfusion of the left liver lobe with Ca²⁺ and Mg²⁺ free HEPES buffer (Invitrogen), followed by HEPES containing 500 mg/l collagenase IV (Sigma-Aldrich). Hepatocytes were separated on a 45% Percoll density gradient (Sigma-Aldrich) and seeded at a density of 50,000 cells/cm² in cell culture plates coated with 0.6 mg/ml rat-tail collagen (Sigma-Aldrich). PMoH were cultured overnight in complete Williams E medium (Invitrogen) containing 10% FBS, 1% glutamine (Sigma-Aldrich), 50μg/ml penicillin/streptomycin (Invitrogen) and 1:1000 gentamicin (Invitrogen). Prior to infection with recombinant adenoviruses, non-adherent cells were washed off and adherent cells incubated with complete Williams E medium supplemented with 1% HEPES pH 7.4 (Invitrogen), 0.1% gentamicin (Invitrogen), 1% glutamine (Invitrogen), 1% linoleic acid (Sigma-Aldrich), 1% epidermal growth factor (EGF) (BD BioScience), 0.1% ITS (Sigma-Aldrich), 0.1% insulin (Sigma-Aldrich), 0.01% dexamethasone (Sigma-Aldrich) and 0.01% ethanolamine (Sigma-Aldrich).

Lim E.J., Chin R., Nachbur U., Silke J., Jia Z., Angus P.W, & Torresi J. (2015). Effect of Immunosuppressive Agents on Hepatocyte Apoptosis Post-Liver Transplantation. PLoS ONE, 10(9), e0138522.

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Isolation of Uncultivable Holospora curviuscula

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Holospora curviuscula has an obligate association with its host, Paramecium bursaria, and is therefore uncultivable, so the bacteria have been grown inside host cells. Holospora curviuscula strain NRB217 from Paramecium bursaria isolated in the Novosibirsk Akademgorodok were obtained from the infected clones maintained in the CCCS (Culture Collection of Ciliates and their Symbionts), Research Park, Saint-Petersburg State University. The host cells were cultivated at the room temperature on the lettuce medium inoculated with Enterobacter aerogenes as a food resource for paramecia. The culture of P. bursaria bearing H. curviuscula was concentrated by centrifugation (10 min at 4500 g) and then homogenized using 10% solution of detergent Nonidet P-40 (Sigma-Aldrich Cat No. 21-3277 SAJ). The infectious forms of H. curviuscula were isolated from the homogenate by centrifugation in Percoll density gradient (Sigma-Aldrich Cat No. P1644) as described previously (Rautian and Wackerow-Kouzova, 2013 (link)). Genomic DNA was isolated with the DNeasy Blood and Tissue kit (QIAGEN Cat No. 69504) using a modified protocol — the time of incubation of cell homogenate with ATL buffer and proteinase K was increased to 16 h. All the subsequent steps were performed according to the standard Quick-Start Protocol.

Garushyants S.K., Beliavskaia A.Y., Malko D.B., Logacheva M.D., Rautian M.S, & Gelfand M.S. (2018). Comparative Genomic Analysis of Holospora spp., Intranuclear Symbionts of Paramecia. Frontiers in Microbiology, 9, 738.

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Isolation of Intestinal Lamina Propria Lymphocytes

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Small or large intestine was mechanically dissected and flushed with ice-cold PBS. The intestines were cut into 8 pieces and incubated in the presence of 1mM DTT and 5mM EDTA at 37^oC for 30 min. The digested pieces were passed through a 100μm cell strainer. The cell suspension was discarded. The remaining pieces were cut into 1mm pieces and further digested in RPMI 1640 medium with collagenase D (1mg/ml collagenase D, Roche), DNase I (100μg/ ml, Sigma), Liberase TL (0.2mg/ml, Roche) and 10% FBS at 37°C for 1h. The LPL were isolated by ce ntrifuging at 2000rpm for 20 min with 40% and 80% discontinuous Percoll density gradient (Sigma). Isolated LPL were then subjected to subsequent analysis.

Wu B., Zhang S., Guo Z., Wang G., Zhang G., Xie L., Lou J., Chen X., Wu D., Bergmeier W., Zheng J, & Wan Y.Y. (2018). RAS P21 Protein Activator 3 (RASA3) specifically promotes pathogenic T helper 17 cell generation by repressing T helper 2-biased programs. Immunity, 49(5), 886-898.e5.

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Isolation and Analysis of Intrahepatic Lymphocytes

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Intrahepatic lymphocytes (IHLs) were isolated as previously described (30 (link)). In brief, liver was perfused with 10 ml PBS, minced and digested with RPMI 1640 containing 0.05% collagenase IV (Roche, Indianapolis, IN) at 37 °C for 30 min. After digestion, cell suspensions were passed through 70 μm cell strainers, followed by a centrifugation over a 30/70% discontinuous Percoll density gradient (Sigma, St. Louis, MO) at 400 g at room temperature for 30 min. The cells were collected from the interphase, washed, resuspended in complete RPMI 1640, and the total number of IHLs per liver was counted. The relative percentages of immune cell populations were then analyzed by flow cytometry, and the absolute numbers of these lymphocyte subpopulations per liver were calculated according to their percentages and total IHL numbers in each liver.

Wang H., Wang G., Banerjee N., Liang Y., Du X., Boor P.J., Hoffman K.L, & Khan M.F. (2021). Aberrant Gut Microbiome Contributes to Intestinal Oxidative Stress, Barrier Dysfunction, Inflammation and Systemic Autoimmune Responses in MRL/lpr Mice. Frontiers in Immunology, 12, 651191.

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In Vitro Bovine Embryo Production

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IVF was performed in TALP medium containing 2 mM penicillamine, 1 mM hypotaurine, 250 mM epinephrine, 20 μg/mL heparin, 114 mM NaCl, 3.2 mM KCl, 0.4 mM NaH₂PO₄, 10 mM sodium lactate, 25 mM NaHCO₃, 0.5 mM MgCl₂–6H₂O, 2.0 mM CaCl₂–2H₂O, 6 mg/mL BSA, 5 μL/mL gentamicin, and 0.2 mM sodium pyruvate. Frozen-thawed semen (at 37°C in water bath for 30 s) from a single proven fertility bull or raw semen of all low-fertility bulls was prepared by Percoll density gradient (Sigma Aldrich) (45/90%). Semen was thawed at 37°C for 30 s, placed on the top of the Percoll gradient, and centrifuged for 30 min at 300 g. The semen suspension was diluted in the appropriate volume of fertilization medium to obtain a final concentration of 10⁷ spermatozoa per milliliters.
An aliquot of 10 µL of semen was co-incubated with matured oocytes for 18 h at 38.5°C in 5% CO_2. Culture was performed in 70 μL droplets (up to 20 oocytes/droplet) of the medium under mineral oil. At the end of gamete co-culture, cumulus cells were completely removed, and cumulus-free presumptive zygotes were randomly transferred into different culture systems and cultured up to day 7.

Lange-Consiglio A., Capra E., Monferini N., Canesi S., Bosi G., Cretich M., Frigerio R., Galbiati V., Bertuzzo F., Cobalchini F., Cremonesi F, & Gasparrini B. (2022). Extracellular vesicles from seminal plasma to improve fertilizing capacity of bulls. Reproduction & Fertility, 3(4), 313-327.

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Isolation of Intrahepatic and Splenic Lymphocytes

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Intrahepatic lymphocytes were isolated following our earlier published method (Wang et al., 2019 (link)). Briefly, the liver was perfused with PBS, minced and digested with RPMI-1640 containing 0.05% collagenase IV (Roche, Indianapolis, IN) at 37 °C for 30 min. Cell suspensions were passed through 70-μm cell strainers, followed by a enrichment centrifugation (400g for 30 min) over a 30/70% discontinuous Percoll density gradient (Sigma) at room temperature. The cells were then collected from the interphase, washed, and resuspended in complete RPMI-1640 containing 10% FBS. The spleens were gently mashed in RPMI-1640 medium through a cell strainer. Red blood cells were removed by using Red Cell Lysis buffer (Sigma). Cells were harvested by centrifugation and resuspended in complete RPMI-1640 containing 10% FBS.

Wang H., Banerjee N., Liang Y., Wang G., Hoffman K.L, & Khan M.F. (2021). Gut microbiome-host interactions in driving environmental pollutant trichloroethene-mediated autoimmunity. Toxicology and applied pharmacology, 424, 115597.

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Spinal Cord Immune Cell Isolation

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Mice were sacrificed and perfused through the heart with 25 ml of PBS using an 18-gauge needle. Spinal cords were flushed out of the spines by inserting a 5-ml syringe needle filled with 1× PBS, minced with dissection scissors, and incubated in 1× HBSS containing 4,000 U/ml collagenase D (Roche) and 10 ng/ml DNase I (Roche) for 30 min at 37°C. Enzymatic digestion was stopped by adding 0.5 M EDTA to a 12.5 mM final concentration, and tissue was pipetted up and down with a Pasteur pipette to release cells. Samples were then filtered through a 70-µm mesh and washed with PBS supplemented with 2% FBS (Atlanta Biologicals) to remove collagenase D, and then pellets were purified using a 30%/70% Percoll density gradient (Sigma) to separate immune cells from myelin. After collection, immune cells were washed twice with PBS, counted, and stained for flow cytometry (antibodies panel described below).

Mimouna S., Rollins D.A., Shibu G., Tharmalingam B., Deochand D.K., Chen X., Oliver D., Chinenov Y, & Rogatsky I. (2020). Transcription cofactor GRIP1 differentially affects myeloid cell–driven neuroinflammation and response to IFN-β therapy. The Journal of Experimental Medicine, 218(1), e20192386.

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Isolation and Characterization of Intrahepatic Lymphocytes

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Intrahepatic lymphocytes (IHLs) were isolated as we described earlier [38 (link)]. Briefly, the liver was perfused with PBS, minced and digested with RPMI 1640 containing 0.05% collagenase IV (Roche, Indianapolis, IN) at 37 °C for 30 min. After digestion, cell suspensions were passed through 70 μm cell strainers, followed by a centrifugation over a 30/70% discontinuous Percoll density gradient (Sigma, St. Louis, MO) at 400 g room temperature (RT) for 30 min. The cells were collected from the interphase, washed, and resuspended in complete RPMI 1640 containing 10% FBS. The total number of IHLs per liver was counted. The relative percentages of immune cell populations were analyzed by flow cytometry, and the absolute numbers of these lymphocyte subpopulations per liver were calculated according to their percentages and the total IHL numbers in each liver.

Wang H., Wang G., Liang Y., Du X., Boor P.J., Sun J, & Khan M.F. (2019). Redox regulation of hepatic NLRP3 inflammasome activation and immune dysregulation in Trichloroethene-mediated autoimmunity. Free radical biology & medicine, 143, 223-231.

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