Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using a Ficoll gradient (Axis Shield, Norway). Unstimulated PBMCs were stained with APC anti-human CD19 antibody (BioLegend cat #302212), APC/Cyanine7 anti-human CD138 antibody (BioLegend cat #356528), PerCP/Cyanine5.5 anti-human CD1d antibody (BioLegend cat #350312), APC anti-human CD25 antibody (BioLegend cat #302610), and PE anti-human CD223 (LAG-3) antibody (BioLegend cat #369306). After surface staining, cells were stained with PE anti-human EBi3 antibody (BioLegend cat #360904) and PE/Cy7 anti-human IL-10 antibody (BioLegend cat #501420), FITC anti-human FOXP3 antibody (BioLegend cat #320106). Corresponding isotype controls were used for each antibody. All cells were analyzed with BD LSR II flow cytometer using DIVA software version 8.0.
Ficoll gradient
Manufactured by Axis-Shield
Sourced in Norway
Ficoll gradient is a density gradient medium used for the separation and purification of cells and subcellular organelles. It is a synthetic, high-molecular-weight, highly branched polymer of sucrose that creates a density gradient when centrifuged. This gradient allows the separation and isolation of different cell types or organelles based on their buoyant density.
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Lab products found in correlation
Lsr 2 flow cytometer, by BD (4 mentions)
Apc anti human cd19 antibody, by BioLegend (2 mentions)
Fitc anti human foxp3 antibody, by BioLegend (2 mentions)
Apc cyanine7 anti human cd138 antibody, by BioLegend (2 mentions)
Percp cyanine5.5 anti human cd1d antibody, by BioLegend (2 mentions)
Apc anti human cd25 antibody, by BioLegend (2 mentions)
Pe anti human cd223 lag 3 antibody, by BioLegend (2 mentions)
Pe anti human ebi3 antibody, by BioLegend (2 mentions)
Pe cy7 anti human il 10 antibody, by BioLegend (2 mentions)
Easysep human b cell enrichment kit, by STEMCELL (1 mentions)
Pe anti mouse cd223 lag 3 antibody, by BioLegend (1 mentions)
Apc anti mouse cd25 antibody, by BioLegend (1 mentions)
Pe cy7 mouse anti human tnfα, by BD (1 mentions)
Cd3 apc, by R&D Systems (1 mentions)
Saponin, by Merck Group (1 mentions)
Facsdiva, by BD (1 mentions)
7 protocols using ficoll gradient
1
Characterization of Immune Cell Subsets in PBMCs
2
B Cell Isolation from Asthmatic Patients
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Peripheral venous blood (20 ml) was drawn from patients and control subjects. Blood was layered over Ficoll gradient (Axis shield, Norway) and centrifuged at 1000 g for 30 minutes. The mononuclear cells layer (PBMC) was then collected and B cells were isolated by negative selection using EasySep Human B cell enrichment kit (StemCell, cat #19054). B cells purity was consistently >98% as evaluated by FACS analysis and the viability of freshly isolated B cells was >99% as evaluated by trypan blue dye exclusion. All experiments were performed using B cells isolated from the same 10 asthmatics and 10 control subjects.
3
Multicolor Flow Cytometry Evaluation of Lymphocyte Subpopulations
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A multicolor flow-cytometry-based approach was used to assess frequency, phenotype and functionality of the major circulating lymphocyte subpopulations and NK cells, before (pre), during (T92) and after treatment (4 and 6 months after enrollment).
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient (Lymphoprep Axis-Shield, Scotland) and frozen as described elsewhere (29 (link)). Samples taken at different time points were tested within the same experimental session, in accordance to “minimal information about T cell (MIATA) and NK (MIANKA) assays” guidelines (http://miataproject.org/miata-guidelines/final-guidelines-2/ ), to improve the data quality level of flow cytometry assay. Cells were thawed in the presence of DNase. Live and dead cells were discriminated by trypan blue exclusion method and samples showing viability less than 70% were not further processed.
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient (Lymphoprep Axis-Shield, Scotland) and frozen as described elsewhere (29 (link)). Samples taken at different time points were tested within the same experimental session, in accordance to “minimal information about T cell (MIATA) and NK (MIANKA) assays” guidelines (
4
Phenotypic Characterization of PBMCs
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Isolation of peripheral blood mononuclear cells (PBMCs) was performed using a Ficoll gradient (Axis-Shield, Norway). The unstimulated PBMCs were stained with APC anti-human CD19 antibody (BioLegend cat #302212), APC/Cyanine7 anti-human CD138 antibody (BioLegend cat #356528), PerCP/Cyanine5.5 anti-human CD1d antibody (BioLegend cat #350312), APC anti-human CD25 antibody (BioLegend cat #302610), and PE anti-human CD223 (LAG-3) antibody (BioLegend cat #369306). Following surface staining, cells were stained with PE anti-human EBi3 antibody (BioLegend cat #360904), PE/Cy7 anti-human IL-10 antibody (BioLegend cat #501420), and FITC anti-human FOXP3 antibody (BioLegend cat #320106). For each antibody, the corresponding isotype controls were used. All cells were analyzed with BD LSR II flow cytometer using DIVA software version 8.0.
5
Lung Lymphocyte Isolation and Characterization
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To isolated lung lymphocytes, mice lungs were collected in complete culture RPMI-1640 medium. Lung tissues were minced gently in complete medium, and the filtrates containing lung lymphocytes and other inflammatory cells were collected. The viability of the freshly isolated cells was evaluated by the Trypan blue dye exclusion test. PBMCs were isolated from blood samples using a Ficoll gradient (Axis Shield, Norway). PBMCs or lung cells were stained with APC anti-mouse CD19 antibody (BioLegend cat # 115512), APC/Cyanine7 anti-mouse CD138 (Syndecan-1) antibody (BioLegend cat # 142530), PerCP/Cyanine5.5 anti-mouse CD1d antibody (BioLegend cat # 123514), APC anti-mouse CD25 antibody (BioLegend cat # 102012), and PE anti-mouse CD223 (LAG-3) antibody (BioLegend cat # 125208). After surface staining, cells were stained with PE anti-mouse IL-27 p28 antibody (BioLegend cat # 516908), PE/Cy7 anti-mouse IL-10 antibody (BioLegend cat # 505026), and FITC anti-mouse FOXP3 antibody. Corresponding isotype controls were used for each antibody. All cells were analyzed with BD LSR II flow cytometer using DIVA software version 8.0. The corresponding data is deposited in S file.
6
Cytokine Expression in PBMCs Exposed to Nanomaterials
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PBMCs were isolated from blood samples of two patients, one with AML and another with CLL, using a Ficoll gradient (Axis Shield, Norway). Cells were collected in complete RPMI 1640 medium (pH 6.5). PBMCs (106 cells) were then divided into five sets for each treatment and incubated with either ZIF-8 (5 μg·ml−1), NV (10 μg·ml−1), or NV-ZIF (5 μg·ml−1) for 1 hour. Cells were then stimulated with PHA (100 ng·ml−1) for 6 or 12 hours (the last 2 hours in the presence of brefeldin A). Intracellular cytokine staining was performed to determine the ability of CD8+ cells to express cytokines. The cells were surface stained with CD3+ APC (0.2 μg·μl−1; R&D Systems, Minneapolis, MN, USA). They were then fixed in 4% paraformaldehyde, resuspended in 0.25% saponin (S4521; Sigma-Aldrich, Germany), and stained with anti–IFN-γ PE-Cy7 [PE-Cy7 mouse anti-human IFN-γ (BD Biosciences), 0.2 μg·μl−1] and anti–TNF-α–PE-Cy7 [PE-Cy7 mouse anti-human TNF-α (BD Biosciences), 0.2 μg·μl−1] antibodies. Samples were analyzed using a BD LSR II flow cytometer equipped with BD FACSDiva (BD Biosciences) software.
7
Isolation and Preservation of Blood Samples
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Blood samples were drawn from each subject in the morning, after overnight fasting and before the dialysis session, using a syringe containing 1.0 mg/mlethylenediaminetetraacetic acid (EDTA) as anticoagulant. Plasma was separated (15 min, 3,0009g, 4 °C), and stored at -80 °C until analysis.
To obtain the peripheral blood mononuclear cells (PBMC), blood samples with EDTA were diluted in phosphate buffered saline (PBS), and cells were separated in 5 ml Ficoll gradient (lymphocyte isolation solution, Axis-Shield, Oslo, Norway) by centrifugation at 800g for 20 min. PBMC were collected and washed twice with cold PBS and separated into two fractions; one of them resuspended in buffer A [10 mM N-(2-hydroxyethyl) piperazine-N 0 -(2-ethanesulfonic acid) (HEPES); pH 7.5, 10 mMKCl, 0.1 mM ethylene glycol tetraacetic acid (EGTA), 0.1 mM EDTA, 1 mMdithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride (PMSF)] for protein analysis, and the second fraction was re-suspended and stored (-80 °C) with 1 ml of recovery cell culture freezing (Invitrogen, Life Technologies, Waltham, MA, USA) for RNA isolation.
To obtain the peripheral blood mononuclear cells (PBMC), blood samples with EDTA were diluted in phosphate buffered saline (PBS), and cells were separated in 5 ml Ficoll gradient (lymphocyte isolation solution, Axis-Shield, Oslo, Norway) by centrifugation at 800g for 20 min. PBMC were collected and washed twice with cold PBS and separated into two fractions; one of them resuspended in buffer A [10 mM N-(2-hydroxyethyl) piperazine-N 0 -(2-ethanesulfonic acid) (HEPES); pH 7.5, 10 mMKCl, 0.1 mM ethylene glycol tetraacetic acid (EGTA), 0.1 mM EDTA, 1 mMdithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride (PMSF)] for protein analysis, and the second fraction was re-suspended and stored (-80 °C) with 1 ml of recovery cell culture freezing (Invitrogen, Life Technologies, Waltham, MA, USA) for RNA isolation.
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