Ficoll hypaque density gradient centrifugation

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Ficoll-Hypaque density gradient centrifugation is a laboratory technique used to separate and isolate different cell types from a heterogeneous mixture. It relies on the differential migration of cells through a density gradient medium during centrifugation. The core function of this method is to facilitate the isolation and purification of specific cell populations based on their unique densities.

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Lab products found in correlation

Anti cd14 beads, by Miltenyi Biotec (1 mentions) Milliplex map human cytokine chemokine kit, by Merck Group (1 mentions) Rpmi 1640 medium, by Sartorius (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)

Close spelling variants of this product

Ficoll (64 citations) Ficoll-Hypaque (30 citations) Ficoll density gradient centrifugation (11 citations) Ficoll gradient (11 citations) Ficoll-Hypaque gradient (5 citations) Ficoll-gradient centrifugation (5 citations) Ficoll-Hypaque density gradient (4 citations) Ficoll density gradient (4 citations) Density gradient centrifugation (2 citations)

5 protocols using ficoll hypaque density gradient centrifugation

Dendritic Cell Differentiation from PBMCs

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Peripheral Blood Mononuclear Cells (PBMCs) were isolated from fresh blood obtained from 5 pediatric CD patients and 6 healthy donors, as controls (from Meyer Children Hospital and the Transfusion Unit of the Careggi Hospital, Florence, Italy, respectively), by Ficoll-Hypaque density gradient centrifugation (Biochrom AG). Monocytes were isolated from low density PBMCs by magnetic enrichment with anti-CD14 beads (Milteny Biotec). Cells were cultured in the presence of GM-CSF (800 U/ml) and recombinant Interleukin [IL-4] (1000 U/ml) for 6 days to allow Dendritic Cells (DC) differentiation as previously described[44 (link)].
All stimulations were carried out by challenging PBMCs with live fungi at 10⁶ cell/ml concentration. After 24 h or 7 days of incubation, supernatants were collected and stored at −20 °C until assayed by means of cytokine detection. Human Milliplex® assay for Tumor Necrosis Factor alpha [TNF-α], Interferon [IFN]-γ IL-1β, IL-6, IL-10, IL-23, IL-12p70 and IL-17A production was performed according to the manufacturer’s instructions using Luminex technology.

Di Paola M., Rizzetto L., Stefanini I., Vitali F., Massi-Benedetti C., Tocci N., Romani L., Ramazzotti M., Lionetti P., De Filippo C, & Cavalieri D. (2020). Comparative immunophenotyping of Saccharomyces cerevisiae and Candida spp. strains from Crohn’s disease patients and their interactions with the gut microbiome. Journal of Translational Autoimmunity, 3, 100036.

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Cytokine Profiling of PBMC-Tumor Cell Co-cultures

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The experimental plan was approved by the local Ethical Committee of Azienda Universitaria Ospedaliera Careggi (AUOC, Careggi Hospital, Florence; Italy), and written informed consent was obtained from all donors (approval document n. 87/10). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coat samples by Ficoll-Hypaque density gradient centrifugation (Biochrom AG). Cells (10⁶ cells/ml) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, at 37°C under 5% CO₂. YL1 or YQ2 cells were added to PBMCs cells at a E:T ratio of 1:10.
Supernatants were collected 5 days later for cytokine detection and conserved at −20°C until assayed. Cytokine detection was performed using the Milliplex^® MAP human cytokine/chemokine kit (Millipore), according to the manufacturer’s instructions.

Cavalieri D., Di Paola M., Rizzetto L., Tocci N., De Filippo C., Lionetti P., Ardizzoni A., Colombari B., Paulone S., Gut I.G., Berná L., Gut M., Blanc J., Kapushesky M., Pericolini E., Blasi E, & Peppoloni S. (2018). Genomic and Phenotypic Variation in Morphogenetic Networks of Two Candida albicans Isolates Subtends Their Different Pathogenic Potential. Frontiers in Immunology, 8, 1997.

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Isolation and Characterization of TILs

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Breast tumor tissues obtained from the surgical specimens were transferred to the Breast Pathology Division. At least 1 cm³ of fresh tumoral tissue was transferred to the Immunology Department and preserved in RPMI-1640 medium (Biological Industries, USA) on ice until the TIL isolation procedure. Fresh tumor samples were minced into ~1 mm³ fragments and transferred into a gentle MACS C tube containing a mix of enzymes H, R, and A (Tumor Dissociation Kit, human; Miltenyi Biotec, Germany). The single-cell suspension was enriched using the gentleMACS™ dissociator (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. TILs were further purified with Ficoll/Hypaque density gradient centrifugation (Biochrom AG, Berlin, Germany). The PD-1, LAG-3, TIM-3, TIGIT, and CTLA-4 expression of CD8⁺ T lymphocytes and NK cell subsets were analyzed using flow cytometry as described below.

Mollavelioglu B., Cetin Aktas E., Cabioglu N., Abbasov A., Onder S., Emiroglu S., Tükenmez M., Muslumanoglu M., Igci A., Deniz G, & Ozmen V. (2022). High co-expression of immune checkpoint receptors PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT on tumor-infiltrating lymphocytes in early-stage breast cancer. World Journal of Surgical Oncology, 20, 349.

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Gastric Cancer Tissue Sample Collection

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All the clinical samples were collected from the First Affiliated Hospital of Nanchang University. In the current study, fresh GC tumor tissues and adjacent normal gastric tissues were collected from patients who underwent surgical resection, blood samples were collected from GC patients diagnosed with primary GC without previously treatment, and paraffin-embedded GC tissues and adjacent noncancerous gastric tissues were obtained from patients who underwent resection between January 2014 and December 2015. Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using Ficoll-Hypaque density gradient centrifugation (Biochrom, Berlin, Germany) according to a previous study [16 (link)]. All of the included patients provided informed consent and signed an agreement. Approval for this study was obtained from the Ethics Committee of the First Affiliated Hospital of Nanchang University.

Fang W., Shi C., Wang Y., Song J, & Zhang L. (2021). microRNA-128-3p inhibits CD4+ regulatory T cells enrichment by targeting interleukin 16 in gastric cancer. Bioengineered, 13(1), 1025-1038.

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Umbilical Cord Blood Mononuclear Cell Isolation and Injection

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Three 50-ml blood samples were collected using sterile collection tubes (50 ml) containing 5 ml citrate phosphate dextrose adenine-1 (CPDA-1) as an anticoagulant immediately after cesarean maternal donor deliveries. Mononuclear stem cells were isolated by Ficoll-Hypaque density gradient centrifugation (Biochrom GmbH, Berlin, Germany) (30 (link)). A volume of 0.2 ml phosphate-buffered saline solution was added to the HUCB MNC pellet for injection of 1 × 10⁶ cells/rat by iv injection into the lateral tail vein using a Hamilton syringe in the control stem cell group, the treated group 24 h after GM treatment, and the preventive group before GM treatment (31 (link)).

Abu Almaaty A.H., Elmasry R.A., Farrag M.S., Althobaiti F., Aldhahrani A., Fayad E, & Hussain M.A. (2021). Impact of Human Umbilical Cord Blood Mononuclear Cells on Gentamicin-Induced Renal Injury and Genotoxicity in Rats. Frontiers in Medicine, 8, 689691.

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