Ficoll paque gradient

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Ficoll-Paque gradient is a separation medium used for the isolation of mononuclear cells from whole blood or bone marrow. It is designed to create a density gradient that allows the separation of different cell types based on their density during centrifugation.

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Ficoll-Paque (1544 citations) Ficoll (495 citations) Ficoll gradient (93 citations) Ficoll-Paque density gradient media (15 citations) Ficoll-Paque density gradient medium (11 citations) Ficoll-Paque gradient separation (6 citations) Ficoll-Paque density gradient solution (5 citations) Ficoll-Paque PREMIUM density gradient media (5 citations) Ficoll®‐Paque PREMIUM density gradient medium (2 citations) Ficoll-Paque PREMIUM gradient solution (2 citations)

90 protocols using ficoll paque gradient

Isolation and Cryopreservation of Tumor and Peripheral Lymphocytes

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Tumor tissue and lymph nodes were cut into pieces of <1 mm³ and placed in a T75 culture flask (Nunc™ EasYFlask™ Cell Culture Flasks, cat. no. 156499, ThermoScientific) with digestion medium, consisting of RPMI (Gibco, Paisley, UK), 10% fetal bovine serum (FBS, Gibco, Paisley, UK), collagenase type IV (1 mg/mL; Gibco, Grand Island, USA), and 12.6 µg/mL recombinant human DNase (Pulmozyme, Roche, Woerden, the Netherlands) for overnight digestion at room temperature. After digestion, the suspension was strained through a 70 µm filter and washed with PBS. Cells were centrifuged over a Ficoll-Paque gradient (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and lymphocytes were isolated from between the two layers. After a wash with PBS, cells were pelleted. Total cell pellet was suspended in 1 ml FBS with 10% dimethylsulfoxide (Merck, Darmstadt, Germany), and stored in liquid nitrogen until further use. Peripheral blood was centrifuged over a Ficoll-Paque gradient (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and PBMC were isolated from between the two layers. After a wash with PBS, cells were pelleted. Total cell pellet was suspended in 1 ml FBS with 10% dimethylsulfoxide (Merck, Darmstadt, Germany), and stored in liquid nitrogen until further use.

Brunekreeft K.L., Paijens S.T., Wouters M.C., Komdeur F.L., Eggink F.A., Lubbers J.M., Workel H.H., Van Der Slikke E.C., Pröpper N.E., Leffers N., Adam J., Pijper H., Plat A., Kol A., Nijman H.W, & De Bruyn M. (2020). Deep immune profiling of ovarian tumors identifies minimal MHC-I expression after neoadjuvant chemotherapy as negatively associated with T-cell-dependent outcome. Oncoimmunology, 9(1), 1760705.

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Isolation of Endothelial Colony-Forming Cells

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Cord blood of seven different donors was used (procedure was approved by the medical research ethics committee, University Medical Center Utrecht, informed consent was obtained from the mothers) as a source for the ECFC isolation (hereafter referred to as ECFC1 to ECFC7; researchers were blinded to the condition of the mother and donor child). Cord blood was diluted with PBS (1:3), and the mononuclear cell layer was retrieved after centrifugation (400 × g) on 1.077 g/ml Ficoll‐Paque gradient (GE Healthcare). 20 × 10⁶ MNCs were plated in a 50‐μg/ml collagen type I‐coated (BD Biosciences, rat tail) well of a six‐well plate with 1 ml of complete endothelial growth medium‐2 (EGM‐2) containing Endothelial Basal Medium‐2 + SingleQuots (Lonza), 100 U/ml‐100 μg/ml PenStrep, and 10% heat‐inactivated FBS. The medium was changed daily until day 7 and then three times per week. Between weeks 2 and 4, ECFC colony outgrowth was observed. When individual colonies expanded, but did not touch each other yet, the cells were trypsinized and replated into collagen type I‐coated culture flasks at a density of ~7,000 cells/cm². Complete EGM‐2 medium was used for subsequent cell expansion. After isolation, ECFCs were either expanded or frozen and used between passages 7 and 12.

Pennings I., van Dijk L.A., van Huuksloot J., Fledderus J.O., Schepers K., Braat A.K., Hsiao E.C., Barruet E., Morales B.M., Verhaar M.C., Rosenberg A.J, & Gawlitta D. (2019). Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures. Journal of Tissue Engineering and Regenerative Medicine, 13(3), 433-445.

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PBMC Isolation and Cryopreservation Protocol

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Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood on Ficoll-Paque gradient (GE Healthcare Life Sciences, USA). After washing in RPMI 1640 medium (Lonza, Switzerland) the cells were counted centrifuged and resuspended in human AB serum (Lonza, Switzerland) containing 10% DMSO for cryoprotection. Cells were aliquoted in cryovials and stored in a –80°C mechanical freezer. Thawing was carried out on the day of fluorescent cell labeling as quickly as possible in 37°C water bath and DMSO (Sigma-Aldrich, Hungary) was washed out twice in RPMI 1640 medium.

Meggyes M., Miko E., Polgar B., Bogar B., Farkas B., Illes Z, & Szereday L. (2014). Peripheral Blood TIM-3 Positive NK and CD8+ T Cells throughout Pregnancy: TIM-3/Galectin-9 Interaction and Its Possible Role during Pregnancy. PLoS ONE, 9(3), e92371.

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Isolation and Cryopreservation of PBMCs

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The isolation of peripheral blood mononuclear cells (PBMCs) has been described earlier [30 (link)]. Briefly, venous blood samples (74 mL) were collected in sodium-heparin collection tubes and the isolation was done by centrifugation over Ficoll-Paque gradient (GE Healthcare Life Sciences, Uppsala, Sweden). PBMCs were subjected to lymphocyte stimulation test (LST) at the concentration ~10x10⁶ and the leftover cells were frozen in 80% Fetal Bovine Serum (FBS, Biowest) and 20% DMSO (Merck, Darmstadt, Germany). Autologous serum obtained from a clotting tube was used as serum in the short-term T-cell proliferation assay as described previously [10 (link),30 (link),31 (link)].

Paaso A., Koskimaa H.M., Welters M.J., Grénman S., Syrjänen K., van der Burg S.H, & Syrjänen S. (2015). Cell mediated immunity against HPV16 E2, E6 and E7 peptides in women with incident CIN and in constantly HPV-negative women followed-up for 10-years. Journal of Translational Medicine, 13, 163.

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Monocyte Migration Assay Protocol

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Human monocytes were isolated as described previously(18 (link), 20 , 29 (link)). Briefly, monocytes were isolated by centrifugation of citrate-phosphate-dextrose-adenine (CPDA)-coagulated blood (S-Monovettes, Sarstedt, Nümbrecht, Germany) on a Ficoll-Paque gradient (GE Healthcare). Leukocytes were cultured overnight at 37°C, 5% CO₂ in a cell culture flask in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FCS (Gibco) and 1% Penicillin/Streptomycin (Sigma-Aldrich, St. Louis, MO, USA). For the migration assay, non-adherent leukocytes were removed and adherent cells were trypsinised (Gibco), centrifuged, and resuspended in medium. Monocyte migration was performed in a modified 48-well Boyden chamber (NeuroProbe Inc., Geithersburg, MD, USA), using a 5 μm pore filter to separate the 2 compartments. Monocytes were added in the upper compartment and 20 μg/ml S100A9 (abcam) or 200 nmol/L CyPA (R&D Systems) or CyPA together with 20 μg/ml of the potential antibodies or _rIgG_2a (Invitrogen, Carlsbad, CA, USA) were added in the lower compartment. The receptor blocking antibody αCD147 and _mIgG₁ were added to the monocytes into the upper compartment. After 4 hours at 37°C, 5% CO₂ the filter was fixed in Methanol, stained in a May-Grünwald/ Giemsa solution (Merck Millipore, Darmstadt, Germany) and the migrated cells on the filter were counted using a microscope.

von Ungern-Sternberg S.N., Vogel S., Walker-Allgaier B., Geue S., Maurer A., Wild A.M., Münzer P., Chatterjee M., Heinzmann D., Kremmer E., Borst O., Loughran P., Zernecke A., Neal M.D., Billiar T.R., Gawaz M, & Seizer P. (2017). Extracellular cyclophilin A augments platelet-dependent thrombosis and thrombo-inflammation. Thrombosis and haemostasis, 117(11), 2063-2078.

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Isolation and Characterization of Plasmablasts

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We sorted single plasmablasts at various time points after either depletion of CD8β+ T cells or interruption of ART. Plasmablasts were defined as CD3⁻ CD20⁻ CD14⁻ CD16⁻ HLA-DR⁺ CD11c⁻ CD123⁻ CD80⁺. Prior to staining, PBMCs were isolated using a Ficoll-Paque gradient (GE Healthcare). Fresh PBMC samples were sorted on a BD FACSJazz sorter (Becton Dickinson). Single plasmablasts were sorted into 96-well plates containing a lysis buffer to preserve the RNA (250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl₂, 6.25 mM dithiothreitol [DTT], and 250 ng/well of yeast tRNA [Life Technologies]; 20 U of RNase inhibitor [New England Biolabs]; and 0.0625 L/well of IGEPAL CA-630 [Sigma]). The RNA plates were immediately frozen and stored at −80°C until subsequent cloning.

Pedreño-Lopez N., Dang C.M., Rosen B.C., Ricciardi M.J., Bailey V.K., Gutman M.J., Gonzalez-Nieto L., Pauthner M.G., Le K., Song G., Andrabi R., Weisgrau K.L., Pomplun N., Martinez-Navio J.M., Fuchs S.P., Wrammert J., Rakasz E.G., Lifson J.D., Martins M.A., Burton D.R., Watkins D.I, & Magnani D.M. (2020). Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies. Molecular Therapy. Methods & Clinical Development, 16, 225-237.

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Isolation and Characterization of H3N2-Reactive Memory B Cells

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Fresh PBMCs were isolated from the collected blood by use of the Ficoll-Paque gradient (GE HealthCare). The CD19⁺/CD27⁺ B cells were stained with biotinylated H3-ATTH and allophycocyanin (APC)-labelled streptavidin. Single H3-reactive memory B cells were excluded doublets using SSC and FSC gate (Supplementary Fig. 10), then were sorted into 384-well plate. After 14 days of expansion, the supernatants were tested for reactivity to recombinant H1 (H1-CA09), H3 (H3-BR07) and H7 (H7-CA444) HA proteins and were analysed by the Meso Scale Discovery multiplex (MSD, Rockville, Maryland). Subsequently, the reactive supernatants were measured in vitro neutralizing activity against H3N2-BR07. All H3N2 neutralizing antibodies were rescued by single-cell RT–PCR using primers as previously described53 (link).

Fu Y., Zhang Z., Sheehan J., Avnir Y., Ridenour C., Sachnik T., Sun J., Hossain M.J., Chen L.M., Zhu Q., Donis R.O, & Marasco W.A. (2016). A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve. Nature Communications, 7, 12780.

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Purification of Tumor-Infiltrating Leukocytes

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TC-1 tumors were harvested at day 20 post implantation, and tumor-infiltrating leucocytes (TILs) were purified using mouse tumor dissociation kit from Miltenyi, following manufacturer’s instructions. Briefly, tumor tissues were chopped into small pieces and resuspended in DMEM medium containing tumor dissociating enzymes (Miltenyi). Tumors were digested on a Gentle MACS with heating system (Miltenyi) using solid tumor program. Enzymatic digestion was stopped by adding cold PBS 0.5% BSA solution and keeping cells on ice. Digested tumors were passed through a 70 μm cell strainer to eliminate remaining undigested tissue. CD45+ cells were purified using CD45 TIL microbeads (Miltenyi) following manufacturer’s protocol. Purified CD45+ cells were used for flow cytometry staining or ex vivo T cell stimulation.
B16-OVA tumor bearing mice were perfused with a saline solution to eliminate blood from the lungs before their collection. Lung-infiltrating leucocytes (LILs) were purified using mouse tumor dissociation kit from Miltenyi, following manufacturer’s instructions.
Peripheral blood and spleen mononuclear cell suspensions from mice were isolated using Ficoll–Paque gradient (GE Healthcare) before flow cytometry analysis, ex vivo stimulation, or TCR avidity assay.

Rossi M., Carboni S., Di Berardino-Besson W., Riva E., Santiago-Raber M.L., Belnoue E, & Derouazi M. (2021). STING Agonist Combined to a Protein-Based Cancer Vaccine Potentiates Peripheral and Intra-Tumoral T Cell Immunity. Frontiers in Immunology, 12, 695056.

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Isolation and Cryopreservation of PBMCs

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The isolation of peripheral blood mononuclear cells (PBMCs) was performed as described in Koskimaa et al., 2014 (link) and Paaso et al., 2015 (link)). Venous blood samples (74 ml) were collected in sodium‐heparin collection tubes. The isolation was performed by centrifugation for 3 h over a Ficoll‐Paque gradient (GE Healthcare Life Sciences, Uppsala, Sweden). Around ~10 × 10⁶ PBMCs were used for the lymphocyte stimulation test (LST). The leftover cells were cryopreserved in 80% Fetal Bovine Serum (FBS, Biowest, EU quality) and 20% DMSO (Merck, Darmstadt, Germany) at a density of 10 million PBMCs/vial. Autologous serum was used for the cell cultures in the short‐term T cell proliferation assay (Koskimaa et al., 2014 (link); Paaso et al., 2015 (link)).

Paaso A., Koskimaa H., Welters M.J., Kero K., Rautava J., Syrjänen K., van der Burg S.H, & Syrjänen S. (2021). Interferon‐γ and IL‐5 associated cell‐mediated immune responses to HPV16 E2 and E6 distinguish between persistent oral HPV16 infections and noninfected mucosa. Clinical and Experimental Dental Research, 7(5), 903-913.

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Neutrophil Isolation and c-Abl Kinase Analysis

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Blood was collected from the inferior vena cava in acid citrate dextrose (1:10). Blood samples were added to Roswell Park Memorial Institute medium 1640 (RPMI 1640, Invitrogen, Stockholm, Sweden) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 2 mM EDTA (Sigma-Aldrich, Stockholm, Sweden). Following hypotonic lysis (5 ml ice-cold 0.2% NaCl, added for 45 s followed by addition of 5 ml 1.6% NaCl), neutrophils were separated from mononuclear cells by density gradient centrifugation using a Ficoll-Paque gradient (GE Healthcare, Uppsala, Sweden). The neutrophil layer was isolated and washed with RPMI 1640 and cells were resuspended at 4 × 10⁶ cells/ml. Next, the cells were homogenized and the activity of c-Abl kinase determined in isolated neutrophils as described below.

Hawez A., Ding Z., Taha D., Madhi R., Rahman M, & Thorlacius H. (2021). c-Abl kinase regulates neutrophil extracellular trap formation and lung injury in abdominal sepsis. Laboratory Investigation; a Journal of Technical Methods and Pathology, 102(3), 263-271.

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