Approximately 40mL of blood was drawn before and after at least 8 weeks post-IRT in heparinized tubes (BD Biosciences, NJ, USA) Blood was processed into peripheral blood mononuclear cells (PBMCs) via Ficoll gradient separation (GE Healthcare, MA, USA) and subsequently cryopreserved at -80°C in 90% FBS + 10% DMSO. PBMCs were later thawed in batches and rested overnight cultured in RP-10 media (RPMI 1640 (Gibco) supplemented with 10% FBS, 50 μM 2-mercaptoethanol (Gibco), 100μg/mL penicillin (Gibco), 100μg/mL streptomycin (Gibco), 10mM HEPES (Gibco), and 10 mM L-glutamine (Wisent, QC, Canada)) before use in assays at 37°C and 5% CO2.
Ficoll gradient separation
Manufactured by GE Healthcare
Sourced in Spain
Ficoll gradient separation is a centrifugation technique used to isolate specific cell populations from complex biological samples. It utilizes a density gradient medium composed of sucrose-epichlorohydrin copolymers to separate different cell types based on their distinct densities. This method allows for the efficient isolation and purification of various cell types, including mononuclear cells, granulocytes, and other cell populations, for further analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Wisent (1 mentions)
Heparinized tube, by BD (1 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Nylon wool column, by Polysciences (1 mentions)
Human macrophage colony stimulating factor m csf, by Miltenyi Biotec (1 mentions)
96 well plate, by Corning (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
9 protocols using ficoll gradient separation
1
PBMC Isolation and Cryopreservation Protocol
2
Antibody-Mediated Cytotoxicity Assay
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Target cells (2 × 104 cells/well) in 96-well plate were incubated with 5 μg/mL of WT-, Remab6-AF, or isotype human IgG for 30 min in culture media at RT, then added to fresh PBMCs prepared from human blood (IRB#2016P000008) by Ficoll-gradient separation (Cat#17-1440-02, GE Healthcare) at indicated effector/target ratio (E:T) for 24 h at 37 °C in CO2 incubator. The released LDH was measured with Pierce™ LDH Cytotoxicity Assay kit (Cat#88954, Thermo Fisher Scientific) following the manufacturer’s instructions. Percent cytotoxicity was calculated for each E:T ratio as below. Target value with PBMCs with isotype IgG indicates a spontaneous PBMC cytotoxicity.
3
Isolation of Spleen and Peyer's Patch Lymphocytes
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Lymphocytes from spleen and Peyer's patches were isolated as previously described16 (link). Briefly, mouse spleen was homogenized and followed by treatment with ACK lysis buffer to lyses red blood cells. The single-cell suspension was collected by passing splenocytes through 70 μm cell strainers (BD Biosciences, Bedford, MA, USA). Then, the cell suspension was incubated in nylon-wool columns (Polysciences, Warrington, PA, USA) at 37 °C for 1 h and the enriched T cells were washed through the columns with complete RPMI-1640. To isolate lymphocytes from Peyer's patches, visible Peyer's patches were collected from ice-cold PBS-flushed small intestines. After homogenization, cells were passed through 70 μm cell strainers and lymphocytes were isolated by Ficoll gradient separation (GE Healthcare).
4
PBMC Isolation from Rodent Blood
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In rats, part of the troncular blood samples from Control and Cold animals at different ages, collected in Corning tubes with EDTA 0.5 M as an anticoagulant, was used to isolate PBMC by OptiPrep gradient separation (Sigma-Aldrich Química, SL, Madrid, Spain) as previously described (Reynés et al., 2015 (link)). In the ferret experiment, PBMC were isolated from blood samples collected using heparin in NaCl (0.9%) as anticoagulant by Ficoll gradient separation according to the manufacturer's instructions (GE Healthcare Bio Sciences, Barcelona, Spain), with some modifications (Reynés et al., 2017 (link)).
5
Isolation and Activation of Primary CD4+ T Cells from Rat Peripheral Blood
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Primary CD4+ T lymphocytes were isolated from the peripheral blood mononuclear cells of SD rats using Ficoll gradient separation (GE Healthcare, Uppsala, Sweden). T lymphocytes were activated in the presence of plate-bound anti-CD3/anti-CD28 antibodies and cultivated under polarization conditions (13 (link), 14 (link)). The purity of the CD4+ T lymphocytes was verified using FACS analysis after their isolation and found to always remain above 90% (Supplement Figure 1 ). Fibroblast-like synoviocytes were isolated by enzymatic digestion of the synovial tissue from the SD rats (15 (link), 16 (link)).
6
PBMC Isolation from Blood
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PBMC were isolated from blood samples by Ficoll gradient separation according to the instructions of the manufacturer (GE Healthcare Bio Sciences, Barcelona, Spain), with some modifications18 (link).
7
Isolation and Differentiation of Human Macrophages
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Peripheral blood mononuclear cells were isolated from human blood after ficoll gradient separation (GE Healthcare) and CD14+ cells were isolated using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell numbers were determined using a Neubauer chamber by Trypan blue staining, and cells were seeded at a density of 1 × 105 cells per well in 96-well plates (Corning). Monocytes were differentiated into macrophage for 4 days in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and 50 ng/ml human macrophage colony-stimulating factor (M-CSF; Miltenyi Biotec). The growth medium was changed to RPMI supplemented with 10% FBS prior to infection. Macrophages were grown at 37 °C with 5% CO2.
8
Isolation and Characterization of PBMC from Infant Blood
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From the age of 2-6 months, blood samples (1•5-2•5 ml) were collected monthly in ad libitum-fed conditions from the saphena vein at the beginning of the light cycle (08.00-10.00 hours), using heparin in NaCl (0•9 %) as anticoagulant. After blood collection, PBMC were isolated by Ficoll gradient separation, according to the instructions indicated by the manufacturer (GE Healthcare Bio Sciences), with some modifications. In brief, the anticoagulanttreated blood was diluted with an equal volume of balanced salt solution, which was prepared by mixing two stock solutions (1/10): solution A (5•5 mM-anhydrous D-glucose, 5 mM-CaCl 2 .2H 2 O, 0•98 mM-MgCl 2 .6H 2 O, 5•4 mM-KCl, 145 mM-Tris) and solution B (140 mM-NaCl). Next, the blood was layered carefully over Ficoll without intermixing (1•5 ml of Ficoll for 2 ml of blood mixed with balanced salt solution) in a centrifuge tub and centrifuged at 900 g for 40 min at 20°C. PBMC, together with platelets, were harvested from the interface between Ficoll and sample layers. This layer was then centrifuged in balanced salt solution at 400 g for 10 min at 20°C to wash PBMC and to remove the platelets. Every month, blood samples were collected from the saphena vein, stored at room temperature for 1 h and then centrifuged at 1000 g for 10 min at 4°C to collect the serum.
9
Isolation and Preservation of Mononuclear Cells
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Mononuclear cells (MNCs) were isolated from peripheral blood or bone marrow using Ficoll gradient separation (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and stored at -80 °C until DNA extraction. DNA was extracted using a QIAamp DNA blood mini-kit (QIAGEN, GmbH Hilden, Germany) according to the manufacturer's instructions.
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