Ficoll histopaque

Manufactured by Merck Group

Sourced in United States, Spain, Germany, Sao Tome and Principe, France, Brazil, United Kingdom

Ficoll-Histopaque is a density gradient medium used for the separation and isolation of mononuclear cells from whole blood or other cell suspensions. It is a sterile, endotoxin-tested solution containing polysucrose and sodium diatrizoate adjusted to a density of 1.077 g/mL.

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Histopaque (690 citations) Ficoll (251 citations) Ficoll-Histopaque 1077-1 (11 citations) Ficoll Histopaque-1077 gradient (11 citations) Ficoll-Paque (Histopaque 1077 (7 citations) Ficoll-Histopaque gradient (5 citations) Ficoll-Histopaque solution (4 citations) Ficoll-Histopaque reagent (4 citations) Ficoll® PM 400 Histopaque®-1077 (3 citations) Ficoll/Histopaque-1077 Hybri Max (2 citations)

184 protocols using ficoll histopaque

Isolation and Cryopreservation of PBMCs

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Whole blood samples underwent density gradient centrifugation using ficoll histopaque (Sigma, St. Louis, MO, USA), which involved layering whole blood samples onto ficoll histopaque in a 1:1 ratio, centrifuging at 25°C for 30 minutes at 800 G, and using a slow brake to prevent disturbance of the gradient. This allowed for isolation of Peripheral Blood Mononuclear Cells (PBMCs). PBMCs were washed twice with a 1x phosphate-buffered saline (PBS) solution and suspended in 1mL cold fetal bovine serum (FBS) for cryopreservation. FBS 20% DMSO (Dimethyl Sulfoxide) was then slowly added to the suspension, until a final concentration of FBS 10% DMSO was reached. This sample was aliquoted into separate vials to be stored at −80°C prior to in vitro infection.

To K., Cao R., Yegiazaryan A., Owens J., Sasaninia K., Vaughn C., Singh M., Truong E., Sathananthan A, & Venketaraman V. (2021). The Effects of Oral Liposomal Glutathione and In Vitro Everolimus in Altering The Immune Responses Against Mycobacterium bovis BCG Strain in Individuals With Type 2 Diabetes. Biomolecular concepts, 12(1), 16-26.

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Isolation and Cryopreservation of PBMCs

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Vitamin D Modulates Immune Response

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The human blood samples were obtained from healthy volunteers of the local French blood agency (Etablissement Français du Sang, EFS, Auvergne-Rhône-Alpes). After full pertinent information, written informed consent was obtained from all blood donors involved in the study as statutory required (article R.1221–5 of the Public Health Code, 12/01/2009 and 11/06/2006 decrees). Ethical review and approval were not required for this study in accordance with the local legislation and institutional requirements.
PBMC were isolated from the buffy-coats by Ficoll-Histopaque (Sigma) density gradient centrifugation. After isolation, PBMC (10⁶ cells/ml) were cultured for 24 h or 48 h in RPMI-1640 complete medium (37 °C, 5% CO₂) with several doses of 25(OH)D (Sigma Aldrich, ref 739,650) (0 ng/ml: control (D0); 25 ng/ml: deficient (D25); 75 ng/ml: physiological (D75); 125 ng/ml: supraphysiological (D125)), stimulated or not by a bacterial agent (extract of Escherichia coli lipopolysaccharides 055B5 (LPS): 100 ng/ml) ref. L6529 (Sigma-Aldrich), or a viral mimetic agent (synthetic double-stranded RNA Poly (I: C) (PIC): 10 µg/ml) ref. P1038 (Sigma-Aldrich).

Aldekwer S., Goncalves-Mendes N., Bingula R., Martinroche G., Lanchais K., Rougé S., Farges M.C., Rossary A., Diab-Assaf M., Vasson M.P, & Talvas J. (2022). 25-Hydroxyvitamin D potentializes extracellular cathelicidin release from human PBMC stimulated ex vivo with either bacterial (LPS) or viral (P: IC) mimetics. Journal of Physiology and Biochemistry, 78(2), 335-342.

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Isolation and Differentiation of Macrophages from PBMCs

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Blood samples of 200 mL were obtained from healthy donors in sterile heparinized tubes. PBMCs were collected by centrifugation over Ficoll-Histopaque (Sigma-Aldrich), according to product instructions. Half of the isolated PBMCs were employed for differentiation of macrophages from monocytes and the other half was cryopreserved in liquid nitrogen after freezing by slow cooling at approximately 1°C/minute using a Nalgene Freezing Container (Thermo Fisher Scientific). Differentiation of peripheral blood monocytes to macrophages was achieved by culture over 7 days, at which time PBMCs recovered from liquid nitrogen by rapidly thawing at 37°C and having ≥ 90% viability [31 (link)] were evaluated in parallel with monocyte-derived macrophages that were obtained from PBMCs of the same donor.

Fernández O.L., Rosales-Chilama M., Sánchez-Hidalgo A., Gómez P., Rebellón-Sánchez D.E., Regli I.B., Díaz-Varela M., Tacchini-Cottier F, & Saravia N.G. (2024). Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis. PLOS Neglected Tropical Diseases, 18(5), e0012156.

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Monocyte Isolation from PBMCs

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PBMCs were isolated from heparinized blood using Ficoll-Histopaque (Sigma, St. Louis, MO) density gradient separation followed by washing twice with PBS. Cell suspension was made in endotoxin-free RPMI 1640 medium (HyClone) supplemented with L-glutamine (Life Technologies), penicillin/streptomycin (Life Technologies), and 10% fetal calf serum (HyClone). Cells were cultured in 12-well plates for further experiments.²⁸ (link)^,²⁹ (link)^,³⁰ (link) CD14 + monocytes were purified from PBMCs by using the MACS monocyte isolation kit (Miltenyi Biotech, San Diego, CA) according to the manufacturer’s instructions. The purity of enriched monocytes was evaluated by flow cytometry using fluorochrome-conjugated CD14 antibody; the purity of monocytes was about 95%.

Talreja J., Peng C, & Samavati L. (2023). MIF modulates p38/ERK phosphorylation via MKP-1 induction in sarcoidosis. iScience, 27(1), 108746.

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PBMC Isolation and RNA Extraction

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The second set of cultures was processed by Ficoll‐Histopaque gradient centrifugation (Sigma‐Aldrich) after incubation for 72 h to isolate PBMCs. Total RNA extraction from PBMCs was performed using the RNeasy Plus Mini kit (Qiagen GmbH) and concentration was assessed using Nanodrop One (Microvolume UV–Vis Spectrophotometer, Thermo Fisher Scientific). For cDNA synthesis, 2 μg of pure mRNA was reverse transcribed in 20 μl final reaction volume using the High‐Capacity cDNA RT kit (Applied Biosystems, Life Technologies) with oligo (dT) primers (Invitrogen, Life Technologies) extra addition to enhancing the RT reaction.

Ruberto S., Santovito A., Simula E.R., Noli M., Manca M.A, & Sechi L.A. (2022). Bisphenols induce human genomic damage and modulate HERVs/env expression. Environmental and Molecular Mutagenesis, 63(6), 275-285.

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Isolation of PBMCs by Ficoll-Histopaque

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PBMC were isolated by Ficoll-Histopaque (Sigma Chemical Co., St Louis, MO)^{27 (link), 28 (link)} and used directly or cryopreserved.

Park J.J., Wong D.K., Wahed A.S., Lee W.M., Feld J.J., Terrault N., Khalili M., Sterling R.K., Kowdley K.V., Bzowej N., Lau D.T., Kim W.R., Smith C., Carithers R.L., Torrey K.W., Keith J.W., Levine D.L., Traum D., Ho S., Valiga M.E., Johnson G.S., Doo E., Lok A.S, & Chang K.M. (2015). HBV-specific and global T-cell dysfunction in chronic hepatitis B. Gastroenterology, 150(3), 684-695.e5.

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PBMC Isolation from Whole Blood

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For PBMC preparation, blood samples were collected in Vacutainer tubes (Becton-Dickenson) with 5 mM EDTA as anticoagulant. Upon receipt at the Nicaraguan National Virology Laboratory, ~5 ml of blood was transferred into a Leucosep tube (Greiner Bio-One) containing 3 ml of Ficoll Histopaque (Sigma) and centrifuged at 500g for 20 minutes at room temperature. The PBMC fraction was collected and transferred to a tube containing 9 ml of PBS with 2% fetal bovine serum (FBS; Denville Scientific) and 1% penicillin/streptomycin (Sigma). Cells were washed and pelleted three times by centrifugation at 500g for 10 minutes and resuspended in RPMI 1640 complete medium (RPMI 1640, 10% FBS, 1% GlutaMAX™, 1% HEPES and 1% penicillin/streptomycin). Before the third wash, cells were counted using a hemocytometer (Sismex XS-1000i). After the third wash, cells were resuspended in cryovials at a concentration of 3 × 10⁶ cells/ml in freezing medium (90% FBS, 10% dimethyl sulfoxide) and were placed in isopropanol containers (Mr. Frosty, Nalgene) at −80°C overnight and transferred to liquid nitrogen for storage (Michlmayr et al., 2017 (link); Zompi et al., 2012 ).

Young E., Carnahan R.H., Andrade D.V., Kose N., Nargi R.S., Fritch E.J., Munt J.E., Doyle M.P., White L., Baric T.J., Stoops M., DeSilva A., Tse L.V., Martinez D.R., Zhu D., Metz S., Wong M.P., Espinosa D.A., Montoya M., Biering S.B., Sukulpolvi-Petty S., Kuan G., Balmaseda A., Diamond M.S., Harris E., Crowe JE J.r, & Baric R.S. (2020). Identification of Dengue Virus Serotype 3 Specific Antigenic Sites Targeted by Neutralizing Human Antibodies. Cell host & microbe, 27(5), 710-724.e7.

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Expansion and Genetic Modification of NK Cells

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Cord blood (CB) units for research were obtained by the MD Anderson Cancer Center CB Bank, under protocols approved by the institutional review board. Healthy human peripheral blood units were sourced from Gulf Coast Regional Blood Center (Houston, TX). CB and peripheral blood mononuclear cells (PBMCs) were isolated by a density-gradient centrifugation (Ficoll-Histopaque; Sigma, St Louis, MO, USA). CD56-positive NK cells were purified using an NK isolation kit (Miltenyi Biotec, Inc., San Diego, CA, USA), and were stimulated with irradiated (100 Gy) uAPCs (feeder cell to NK cell ratio of 2:1) and recombinant human IL-2 (Proleukin, 200 U/ml; Chiron, Emeryville, CA, USA) in complete serum-free stem cell growth medium (SCGM) (CellGenix GmbH, Freiburg, Germany) on day 0. In specific comparative experiments, uAPC was substituted with C9/IL-21 (15 (link)), a previously characterized feeder cell also harboring mbIL21. Activated NK cells were transduced with retroviral supernatants on day +6 in human fibronectin-coated plates (Clontech Laboratories, Inc., Mountain View, CA, USA). Three days later (on day +9), NK cells were stimulated again with irradiated uAPC and IL-2. On day +15, CAR-transduced NK cells were collected for use in the indicated assays.

Liu E., Ang S.O., Kerbauy L., Basar R., Kaur I., Kaplan M., Li L., Tong Y., Daher M., Ensley E.L., Uprety N., Nunez Cortes A.K., Yang R.Z., Li Y., Shaim H., Reyes Silva F., Lin P., Mohanty V., Acharya S., Shanley M., Muniz-Feliciano L., Banerjee P.P., Chen K., Champlin R.E., Shpall E.J, & Rezvani K. (2021). GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells. Frontiers in Immunology, 12, 626098.

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Expansion and Transduction of CAR-Expressing NK Cells

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CB units for research were provided by the MDACC CB Bank and peripheral blood mononuclear cells (PBMCs) were collected from CLL patients following informed consent, under IRB-approved protocols. CB and PBMCs were isolated by a density-gradient technique (Ficoll-Histopaque; Sigma). CD56+ NK cells, purified with an NK isolation kit (Miltenyi Biotec, Inc., San Diego, CA), were stimulated with irradiated (100 Gy) Clone 9 (2:1 feeder cell:NK ratio) and recombinant human IL-2 (Proleukin, 200 U/mL; Chiron, Emeryville, CA) in complete Serum-free Stem Cell Growth Medium (SCGM) (CellGenix GmbH, Freiburg, Germany) on day 0. Activated NK cells were transduced with retroviral supernatants on day +4 in human fibronectin-coated plates (Clontech Laboratories, Inc., Mountain View, CA). Five days later (day +9), NK cells were stimulated again with irradiated Clone 9 and IL-2. On day +14, CAR-transduced NK cells were harvested for use.

Liu E., Tong Y., Dotti G., Shaim H., Savoldo B., Mukherjee M., Orange J., Wan X., Lu X., Reynolds A., Gagea M., Banerjee P., Cai R., Bdaiwi M.H., Basar R., Muftuoglu M., Li L., Marin D., Wierda W., Keating M., Champlin R., Shpall E, & Rezvani K. (2017). Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent anti-tumor activity. Leukemia, 32(2), 520-531.

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