Biocoll gradient

Manufactured by Harvard Bioscience

Sourced in Germany

Biocoll gradient is a specialized laboratory equipment used for the separation and purification of biological samples. It is designed to create a density gradient, allowing the efficient separation of various cellular components, proteins, or other biomolecules based on their density differences.

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Lab products found in correlation

Neutral protease, by Serva Electrophoresis (3 mentions) Pulmozyme, by Roche (2 mentions) Collagenase nb1, by Serva Electrophoresis (2 mentions) Dnase, by Roche (2 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions) Glutamax 1, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Ficoll paque, by Harvard Bioscience (1 mentions) Phytohaemagglutinin, by Merck Group (1 mentions) Easysep kit, by STEMCELL (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Collagenase, by Serva Electrophoresis (1 mentions) Negative isolation kit, by Miltenyi Biotec (1 mentions) Penicillin, by Harvard Bioscience (1 mentions) Streptomycin, by Harvard Bioscience (1 mentions) Fg0415, by Harvard Bioscience (1 mentions) Human recombinant fgf2, by Thermo Fisher Scientific (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Trucount, by BD (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)

13 protocols using biocoll gradient

Generation of Polyclonal Cytotoxic T Cells

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The murine lymphoblastoid T cell line EL4 was cultured in RPMI 1640 + GlutaMAX-I + 25 mM HEPES (Gibco-Invitrogen) supplemented with 10% foetal bovine serum (Hyclone).
Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, obtained from healthy donors, by separation on a Biocoll gradient (Biochrom) according to standard procedures. To generate polyclonal cytotoxic T cells, CD8⁺ T cells were purified using MojoSort purification kit (Biolegend) and activated with anti-CD3/CD28 coated plates (5 and 3 μg/ml respectively). After two days, media was supplemented with IL-2 (50 U/ml) every 2 days for a time period of 8 days. These studies were performed according to the principles of the Declaration of Helsinki and approved by the local Ethics Committee for Basic Research at the Hospital La Princesa (Madrid); informed consent was obtained from all human volunteers.

Bustos-Morán E., Blas-Rus N., Alcaraz-Serna A., Iborra S., González-Martínez J., Malumbres M, & Sánchez-Madrid F. (2019). Aurora A controls CD8+ T cell cytotoxic activity and antiviral response. Scientific Reports, 9, 2211.

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Standardized PCC Assay Protocol

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Taking into consideration that the PCC assay is technically demanding and not yet widely used, a detailed protocol for PEG mediated cell fusion and PCC induction in lymphocytes isolated from the peripheral blood using mitotic CHO cells was established by the L1 laboratory and shared with the RENEB partners, in order to harmonize and standardize the PCC assay as well as the scoring criteria. Informed consent was obtained for each blood donor, and packaging, labelling and shipment of blood samples conformed to national and international regulations. A temperature logger, dosimeter to record the temperature and any dose received by the samples during transport, was used together with the standardized sample instruction sheet (ISO21243, 2008 , ISO19238, 2014 ). The peripheral blood was always sampled in heparinized vials and, for the isolation of lymphocytes, whole blood was carefully layered on top of equal amount of Ficoll-Paque or Biocoll gradient (Biochrom) in a test tube before centrifugation. In order to quantitate radiation exposure by means of initially induced PCC fragments, the PCC methodology was applied as soon as possible, since the initial radiation-induced fragments in excess of 46 PCCs decrease with time (Pantelias and Maillie, 1985a (link), Darroudi et al., 1998a (link)).

Terzoudi G.I., Pantelias G., Darroudi F., Barszczewska K., Buraczewska I., Depuydt J., Georgieva D., Hadjidekova V., Hatzi V.I., Karachristou I., Karakosta M., Meschini R., M’Kacher R., Montoro A., Palitti F., Pantelias A., Pepe G., Ricoul M., Sabatier L., Sebastià N., Sommer S., Vral A., Zafiropoulos D, & Wojcik A. (2016). Dose assessment inter-comparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay). International journal of radiation biology, 93(1), 48-57.

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Isolation and Stimulation of CD4+ T Lymphoblasts

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For isolation of CD4⁺ T lymphoblast cultures, human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors provided by “Centro de Transfusiones de la Comunidad de Madrid” under an agreement with the Hospital Princesa (Madrid) and approved by the CEIm of Hospital Princesa, according to government ethical consent. Upon separation on a Biocoll gradient (Biochrom, L6115), nonadherent cells were collected after plating PBMCs at 37°C and purified using Stem Cell Technologies EasySep kit for CD4⁺ T cells. Cells were then cultured for 48 h in the presence of SEE (0.01 μg/ml; Toxin Technology) and PHA (0.2 μg/ml phytohaemagglutinin, Sigma Aldrich) to induce lymphocyte proliferation, and IL-2 (50 U/ml) was added to the culture medium every 2 days. HuCD4⁺ T lymphoblasts were transfected 7 days after isolation. Experiments were performed 48 h post-transfection.
Jurkat E6-1 cell line (Vαl.2 Vβ8⁺ TCR) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. A. Weiss. Jurkat E6-1 and Raji lymphoblastoid B cell line were grown in RPMI 1640 medium (Gibco-invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). Cells were cultured at 37 °C, in 5% CO₂ atmosphere. Jurkat E6-1 T cell clones were cultivated with 0.5 mg/mL^-1 G418.

Gómez-Morón Á., Requena S., Pertusa C., Lozano-Prieto M., Calzada-Fraile D., Scagnetti C., Sánchez-García I., Calero-García A.A., Izquierdo M, & Martín-Cófreces N.B. (2023). End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network. Frontiers in Immunology, 14, 1197289.

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Isolation and Purification of Human Islets

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Human islets were obtained through the TU Dresden Islet Transplantation Program approved by the TU Dresden Institutional Review Board (EK 255062022) with written informed consent obtained from each islet donor participant. Islets were isolated and purified from resected pancreas tissue according to a modified Ricordi method. Briefly, collagenase, neutral protease (Serva Electrophoresis, Heidelberg, Germany), and Pulmozyme (Roche, Grenzach, Germany) were infused into the main pancreatic duct. Islets were separated from exocrine tissue by centrifugation on a continuous Biocoll gradient (Biochrom AG, Berlin, Germany) in a COBE 2991 cell processor (Lakewood, CO).

Jarc L., Bandral M., Zanfrini E., Lesche M., Kufrin V., Sendra R., Pezzolla D., Giannios I., Khattak S., Neumann K., Ludwig B, & Gavalas A. (2024). Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions. eLife, 12, RP89962.

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Monocyte Isolation and Macrophage Differentiation

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Human primary monocytes were isolated and purified from peripheral blood mononuclear cells (PBMCs) of buffy coats obtained from healthy blood donors using a Biocoll gradient (Biochrom, Berlin, Germany). After isolation, PBMCs were resuspended in RPMI medium and seeded at a density of 2 × 10⁶ cells/well in non-coated 12-well plates. The monocytes were obtained by selective adherence after 1 h of incubation and cultured over 7 days in RPMI medium supplemented with 10% FBS to allow the differentiation into monocyte-derived macrophages (MDM).

Silva I., Peccerella T., Mueller S, & Rausch V. (2019). IL-1 beta-mediated macrophage-hepatocyte crosstalk upregulates hepcidin under physiological low oxygen levels. Redox Biology, 24, 101209.

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Isolation and Culture of Human Islets

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Human islets of Langerhans were isolated from resected pancreas tissue with appropriate consent and ethical approval at the University Hospital Carl Gustav Carus Dresden according to a modified Ricordi method^{47 (link),48 (link)}. Briefly, Collagenase, neutral protease (Nordmark), and Pulmozyme (Roche) were infused into the main pancreatic duct. Islets were separated from exocrine tissue by centrifugation on a continuous Biocoll gradient (Biochrom AG) in a COBE 2991 cell processor (Lakewood). Following isolation, islets were cultured in RPMI 1640 supplemented with 5.5 mM glucose, 20 mM Hepes, 10% FBS, 0.1% penicillin/streptomycin.

Steenblock C., Richter S., Berger I., Barovic M., Schmid J., Schubert U., Jarzebska N., von Mässenhausen A., Linkermann A., Schürmann A., Pablik J., Dienemann T., Evert K., Rodionov R.N., Semenova N.Y., Zinserling V.A., Gainetdinov R.R., Baretton G., Lindemann D., Solimena M., Ludwig B, & Bornstein S.R. (2021). Viral infiltration of pancreatic islets in patients with COVID-19. Nature Communications, 12, 3534.

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Isolation and Activation of CD4 T Cells

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For isolation of CD4 T cells or T lymphoblast cultures, human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors [provided by “Centro de Transfusiones de la Comunidad de Madrid” under an agreement with the Hospital Princesa (Madrid) and approved by the CEIm of Hospital Princesa, according to government ethical consent] through separation on a Biocoll gradient (Biochrom). Nonadherent cells were collected after plating PBMCs at 37°C. CD4 T cells for synchrotron experiments were isolated with a Miltenyi Biotec negative isolation kit or with a STEMCELL negative isolation kit and used for subsequent procedures. Peripheral blood lymphocytes obtained after adhesion steps were cultured for 48 hours in the presence of SEE (0.01 μg/ml; Toxin Technology) to induce lymphocyte proliferation, and IL-2 (50 U/ml) was added to the culture medium every 2 days until use. Further information and requests for resources and reagents should be directed to and will be fulfilled by corresponding authors.

Martin-Cofreces N.B., Chichon F.J., Calvo E., Torralba D., Bustos-Moran E., Dosil S.G., Rojas-Gomez A., Bonzon-Kulichenko E., Lopez J.A., Otón J., Sorrentino A., Zabala J.C., Vernos I., Vazquez J., Valpuesta J.M, & Sanchez-Madrid F. (2020). The chaperonin CCT controls T cell receptor–driven 3D configuration of centrioles. Science Advances, 6(49), eabb7242.

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Isolation of Human Pancreatic Acinar Cells

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Human acinar cells were obtained from cadaveric donors as a byproduct of pancreatic islet isolation conducted at the University of Wisconsin Department of Surgery, Islet Research Laboratory. Human pancreata were obtained through the University of Wisconsin Hospital and Clinics Organ Procurement Organization with consent obtained for research from next of kin and authorization by the Health Sciences institutional review board. Islets were isolated using a modification of the automated Ricordi method.²¹ (link) Collagenase NB1, neutral protease (Serva Electrophoresis, Heidelberg, Germany), and DNase (Roche Applied Science, Indianapolis, IN) were infused into the main pancreatic duct. Islets were separated from acinar tissue by centrifugation on a continuous Biocoll gradient (Biochrom, Berlin, Germany) in a COBE 2991 cell processor (Lakewood, CO). Acinar cells were washed in DMEM and further purified by allowing to pellet by gravity though DMEM containing 4% bovine serum albumin. Cells were treated as described for rodent acini.

Messenger S.W., Thomas D.D., Cooley M.M., Jones E.K., Falkowski M.A., August B.K., Fernandez L.A., Gorelick F.S, & Groblewski G.E. (2015). Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells. Cellular and Molecular Gastroenterology and Hepatology, 1(6), 695-709.

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Human Bone Marrow-Derived MSC Isolation

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Human bone marrow-derived MSCs were isolated from human bone marrow aspirates of two different donors. Donor A was a 22-year old male and donor B was a 23-year old female. Mononuclear cells were isolated by density gradient centrifugation with a Biocoll gradient (Biochrom). Plastic-adherent cells were passaged before reaching confluence and were sub-cultured at a density of 2 x 10³ to 5 x 10³ cells/cm² in Dulbecco´s Modified Eagle´s Medium (DMEM, FG0415, Biochrom) supplemented with 10% fetal calf serum (FCS, Thermo Scientific, Hyclone, Waltham, USA), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Life Technologies), 100 U/mL penicillin (Biochrom), 100 mg/mL streptomycin (Biochrom) and 2 ng/mL human recombinant FGF2 (Peprotech, Hamburg, Germany). For all experiments, cells were used in passage 6. MSCs characteristics were confirmed randomly by flow cytometric analysis of cell surface molecules and by in vitro differentiation as described elsewhere [38 (link),39 (link)].

Schulze J., Kaiser O., Paasche G., Lamm H., Pich A., Hoffmann A., Lenarz T, & Warnecke A. (2017). Effect of hyperbaric oxygen on BDNF-release and neuroprotection: Investigations with human mesenchymal stem cells and genetically modified NIH3T3 fibroblasts as putative cell therapeutics. PLoS ONE, 12(5), e0178182.

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Isolation of Human Pancreatic Acinar Cells

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Human acinar cells were obtained from cadaveric donors as a byproduct of pancreatic islet isolation conducted at the UW Department of Surgery, Islet Research Lab. Human pancreata were obtained through the University of Wisconsin Hospital and Clinics Organ Procurement Organization with consent obtained for research from next of kin and authorization by the Health Sciences Institutional Review Board. Islets were isolated using a modification of the automated Ricordi method ^{21 (link)}. Collagenase NB1, neutral protease (Serva Electrophoresis, Heidelberg, Germany), and DNase (Roche, Indianapolis, IN) were infused into the main pancreatic duct. Islets were separated from acinar tissue by centrifugation on a continuous Biocoll gradient (Biochrom AG, Berlin, Germany) in a COBE 2991 cell processor (Lakewood, CO). Acinar cells were washed in DMEM and further purified by allowing to pellet by gravity though DMEM containing 4% BSA. Cells were treated as described for rodent acini.

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