Heparinized fresh whole blood was obtained from healthy donors of the NIH Clinical Center Department of Transfusion Medicine (n= 32) after informed consent was obtained in accordance with the Declaration of Helsinki [32 (link)] (NIH, Bethesda, MD, USA). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque gradient centrifugation (GE Healthcare, Chicago, IL) with Leucosep centrifuge tubes (VWR, Radnor, PA), according to manufacturer’s instructions. Cells were frozen in medium containing 90% FCS and 10% dymethyl-sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and stored in liquid nitrogen until use.
Ficoll paque gradient centrifugation
Manufactured by GE Healthcare
Sourced in United Kingdom, Sweden, United States
Ficoll-Paque gradient centrifugation is a laboratory technique used for the separation and purification of cells, particles, and macromolecules based on their density differences. It involves the use of a density gradient medium, Ficoll-Paque, which allows the selective separation of different cell types or molecules during centrifugation.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (2 mentions)
Leucosep centrifuge tubes, by Avantor (1 mentions)
Negative selection kit, by Miltenyi Biotec (1 mentions)
Positive selection microbeads, by STEMCELL (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
70 μm strainer, by BD (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Percoll gradient, by Merck Group (1 mentions)
Cellquest pro software, by BD (1 mentions)
Immunomagnetic bead selection, by STEMCELL (1 mentions)
75 cm2 culture flask, by Thermo Fisher Scientific (1 mentions)
Transferrin, by Lonza (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Gm csf, by Gentaur (1 mentions)
Interleukin 4 (il 4), by Gentaur (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Vacuette edta tubes, by Greiner (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
13 protocols using ficoll paque gradient centrifugation
1
PBMC Isolation and Cryopreservation
2
Isolating and Profiling Memory T Cells in CF
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Peripheral blood samples were collected from adult CF patients attending the West of Scotland Adult CF Unit, Glasgow. Healthy individuals (controls) had no history of respiratory disease or inter-current illness. Participant characteristics are shown in Table 1 . Patients with CF attending the West of Scotland Adult CF Unit are deemed to be chronically colonized with Pseudomonas aeruginosa if sputum cultures remain positive for the organism after two attempts to clear the organism with combination antibiotic eradication therapy. Intermittent infection is deemed to exist when PA has been isolated and eradicated via antibiotic therapy.
PBMCs were obtained by Ficoll-Paque gradient centrifugation (GE Healthcare). CD14+ monocytes were then magnetically isolated by a positive selection kit (Miltenyi Biotech). Memory CD4+ T cells (purity for CD4+CD45RO+ >98%) were magnetically isolated by a negative selection kit (Miltenyi Biotech). Memory CD4+ T cells were further sorted into CCR6-enriched and CCR6-depleted populations using positive selection microbeads (Stemcell Technologies). Proliferation was measured with CFSE (carboxyfluorescein diacetate succinimidyl ester; Invitrogen) or proliferation dye eFluor®450 (eBioscience) incorporated prior to cell culture.
PBMCs were obtained by Ficoll-Paque gradient centrifugation (GE Healthcare). CD14+ monocytes were then magnetically isolated by a positive selection kit (Miltenyi Biotech). Memory CD4+ T cells (purity for CD4+CD45RO+ >98%) were magnetically isolated by a negative selection kit (Miltenyi Biotech). Memory CD4+ T cells were further sorted into CCR6-enriched and CCR6-depleted populations using positive selection microbeads (Stemcell Technologies). Proliferation was measured with CFSE (carboxyfluorescein diacetate succinimidyl ester; Invitrogen) or proliferation dye eFluor®450 (eBioscience) incorporated prior to cell culture.
3
Immunophenotypic Characterization of CLL
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Detailed evaluation employed samples from 8 patients, confirmatory study of morphology and of further biochemical aspects used samples from 10 additional patients. Patient samples were collected with informed consent with approval of the local Research Ethics Committee (reference 10/H1017/73). All samples had typical clinical presentation and immunophenotype with no adverse cytogenetic features, and comprised >95% CLL B-lymphocytes as determined by flow cytometry. Specific samples differing in the presence or absence of somatic mutation of IgVH genes were kindly provided from the Leukaemia Lymphoma Research -Cell Bank Facility at the Royal Liverpool University Hospital. Tissues from diagnostic use were employed to examine cell interactions within tissue sections. Mononuclear cells were isolated by Ficoll-Paque gradient centrifugation (GE Healthcare, Buckinghamshire, UK) and were used freshly or were cryopreserved until use in 90% fetal bovine serum, 10% DMSO cells.
4
Evaluating Cell Viability in MM Cells
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For cell viability assays, 3000 cells were plated in sterile 96-well plates and cultured overnight. Compounds were then added in serial dilutions. The Bay 11–7084 was purchased from Santa Cruz (CAT# sc-202490). Cellular viability was determined after 48 hours incubation by the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Plates were measured on a THERMO max microplate reader. For stromal co-culture cell assay, BMSCs were obtained from bone marrow of MM patients. Briefly, bone marrow aspirates were subjected to Ficoll-Paque gradient centrifugation (GE Healthcare, Little Chalfont, United Kingdom), and mononuclear cells (MNCs) were collected and expanded in human complete MesenCult medium (STEMCELL Technologies, Canada) for 2 weeks. Then a confluent monolayer was generated by plating 10×105 BMSCs in a 96-well plate for additional 48 hours. MM.1S cells were treated with Bortezomib and plated in co-culture with BMSCs for 48 hours at 37°C. Cell Viability was assessed by the CellTiter-Glo Luminescent intensity.
5
Isolation and Analysis of Splenic Regulatory T Cells
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Mouse spleens and kidneys were cut into pieces, milled with a tissue grinder, and filtered using a 70-μm strainer (BD FALCON, USA). Splenocytes were isolated by Ficoll-Paque gradient centrifugation (GE Healthcare, Sweden). Mononuclear spleen cells (1 × 106 cells/well) were cultured with IL-2 (10 pg/mL) (BD PHARMINGEN) in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum for 6 days and analyzed by flow cytometry.
Splenic cells were incubated with magnetic beads coated with an antibody against CD4 using a commercial magnetic-activated cell sorting (MACS) kit (Mitenyi Biotec). FOXP3+IL-17+ cells were subsequently sorted by fluorescence-activated cell sorting (FACS) (purity >95%) and checked by flow cytometry (FC500, Beckman Coulter, Fullerton, CA).
Splenic cells were incubated with magnetic beads coated with an antibody against CD4 using a commercial magnetic-activated cell sorting (MACS) kit (Mitenyi Biotec). FOXP3+IL-17+ cells were subsequently sorted by fluorescence-activated cell sorting (FACS) (purity >95%) and checked by flow cytometry (FC500, Beckman Coulter, Fullerton, CA).
6
Isolation of Resting B Cells from Tonsils
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Tonsils of children undergoing routine tonsillectomies at Saint István Hospital were used in compliance with the Declaration of Helsinki. The study was approved by the Ethics Committee of the Medical Research Council in Hungary (TUKEB; 52088/2015/EKU). Mononuclear cells were isolated from the tonsils by applying Ficoll-Paque gradient centrifugation (GE Healthcare, Chicago, IL, USA). T cells were removed by rosetting mononuclear cells with 2-aminoethylisothiouronium bromide (AET; Sigma-Aldrich, St. Louis, MO, USA) -treated sheep red blood cells using Ficoll-Paque centrifugation. Resting B cells were obtained by centrifugation on Percoll gradient (Sigma-Aldrich, St. Louis, MO, USA). The isolation efficiency yielded above 95% in each case, which was verified by using a FACSCalibur flow cytometer (Becton-Dickinson Biosciences, San Jose, CA, USA), through FITC-conjugated mouse anti-human CD19 monoclonal antibody staining (ImmunoTools, Friesoythe, Germany) for 30 min at 4°C and using CellQuest Pro software (Becton-Dickinson Biosciences).
7
Profiling Multiple Myeloma Transcriptomes
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For gene expression profiling and evaluation of genomic variants in the Institute for Molecular Medicine Finland (FIMM) cohort, bone marrow aspirates were obtained from multiple myeloma patients after obtaining written informed consent and following protocols approved by an ethical committee of the Helsinki University Hospital Comprehensive Cancer Center, and in compliance with the Declaration of Helsinki. Matched patient skin biopsies were collected (also with informed consent) at the same time and from the same site as bone marrow aspirates, and in accordance with approved protocols, for constitutional DNA analysis. Bone marrow mononuclear cells were isolated by Ficoll-Paque gradient centrifugation (GE Healthcare), and CD138+ plasma cells enriched by immuno-magnetic bead selection (StemCell Technologies).
8
Ex vivo PBMC Cytokine Induction
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After a maximum storage period of 1 h of freshly collected EDTA blood at room temperature, PBMC were purified using Ficoll-Paque gradient centrifugation (GE healthcare Life Sciences, NJ, USA). PBMC (1 × 106/mL) was then incubated with or without NOD2 ligand muramyl dipeptide (MDP, 1 μg/mL) (Invivogen Corp, San Diego, CA, USA) for 24 h at 37 °C in a 5% CO2 atmosphere. After incubation, the cell free supernatant of ex vivo culture was harvested and stored at −70 °C for subsequent assay of inflammatory cytokines using human inflammatory cytokine cytometric bead array (CBA) kit with the FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) [50 (link)]. The % increase of the cytokine induction by PGN and MDP was calculated by (concentration of cytokines stimulated by ligand − basal concentration of cytokines without any stimulation)/basal concentration of cytokine without any stimulation × 100%.
9
Isolation and Cryopreservation of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated within 24 hours of collection from whole blood using Ficoll-Paque gradient centrifugation (GE Healthcare, Chicago, IL), cryopreserved and stored in 90% FBS containing 10% DMSO in liquid nitrogen until further use. HIV-1 seronegative donor blood cells obtained from New York Blood Center (New York City, NY) were used as controls to optimize the antibody concentrations for the multiparameter flow cytometry panel (Supplementary Table 1
10
Generation of DC-based Lung Cancer Vaccine
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Peripheral blood mononuclear cells (PBMC) were obtained from buffy coats of healthy donors by Ficoll-Paque gradient centrifugation (GE Healthcare). 75 × 106 PBMCs/flask in 10 ml of serum free GMP-compliant media X-VIVO 15 (supplemented with transferrin, Lonza) were plated in 75 cm2 culture flasks (Nunc) at 37°C and monocytes were allowed to adhere to the flask bottom for 2 h. The non-adherent fraction containing lymphocytes was collected and stored short-term at -80°C. Monocyte-derived DC were generated in X-VIVO 15 for 4 days [26 (link)] in the presence of 500 IU/ml of GM-CSF and 20 ng/ml of IL-4 (both from Gentaur). To generate DC-based HHP lung cancer vaccine, 2 × 105 immature DC/well seeded in 96-well plate (Nunc) were incubated with an equal mixture (1 H520:1 H522) of thawed HHP-killed lung cancer cell lines at a DC/tumor cells ratio of 5:1 for 4 h. After that poly(I:C) (25 μg/ml, VacciGradeTM InvivoGen) was added for additional 20 h.
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