Human blood from six anonymous individuals was purchased from the New York Blood Bank (New York City, NY). Since the blood samples from the “Blood Bank” are deidentified samples, this project is exempted from human subject research. PBMCs were isolated by gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Sweden) [65 (link)–66 (link)]. In brief, peripheral blood from healthy volunteers was diluted with an equal volume of sterile phosphate-buffered saline (PBS; Gibco by life Technologies) and slowly layered onto the Ficoll-Paque media solution. Tubes were centrifuged at 500g for 30 to 40 min at 18 °C. After centrifugation a thin layer of mononuclear cells could be detected between plasma and Ficoll-Paque media. The layer of mononuclear cells was transferred to a sterile tube and was washed with sterile PBS three times. After the last wash, cells were resuspended in RPMI1640, supplemented with 10% fetal bovine serum and counted using a hemocytometer. This method led to more than 97% pure and live cells. Freshly isolated PBMCs were adjusted to 2.5 × 105 in 24 well-plates (Costar, ME, USA) for cell culture and bio-uptake experiments, and 2.5 × 104 cells in 96 well-plates (Corning Incorporated, NY, USA) for cell viability and metabolic activity test.
Ficoll paque
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Ficoll-Paque is a density gradient medium used for the isolation and purification of cells, organelles, and other biological macromolecules. It is a sterile, apyrogenic, and endotoxin-tested solution that provides a gentle, non-toxic environment for sensitive cells during separation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Phosphate buffered saline (pbs), by Merck Group (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Ficoll paque, by GE Healthcare (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Dfc295, by Leica (3 mentions)
Dm il led, by Leica (3 mentions)
Molecular imaging software v 4.5.1 imaging system, by Kodak (2 mentions)
Rneasy mini kit isolation kit, by Qiagen (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Tgf β, by Thermo Fisher Scientific (2 mentions)
Tissue culture flask, by Corning (2 mentions)
β carotene, by Merck Group (2 mentions)
Macs monocyte isolation kit, by Miltenyi Biotec (2 mentions)
24 well plate, by Corning (1 mentions)
96 well plate, by Corning (1 mentions)
59 protocols using ficoll paque
1
PBMC Isolation from Whole Blood
2
Isolation and Culture of Human PBMCs
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Human peripheral blood mononuclear cells (PBMCs) were donated from healthy volunteers and purified by centrifugation on Ficoll-Hypaque PLUS (GE Healthcare Life Science, Pittsburgh, PA, USA), as previously described.5 (link) Briefly, PBMCs were isolated by Ficoll-Paque and washed three times with DPBS (Gibco). The purified T cells were maintained in complete culture medium supplemented with 10% fetal bovine serum and the mixed lymphocyte reaction (MLR) was undertaken within a week. The ‘T cells’ described in this section were human PBMCs, and these cells were used as the sources of enriched responder lymphocytes. For positive controls, T cells were treated with PHA (5 μg ml−1; Sigma, Irvine KA, MO, USA) and/or allogeneic T cells. All volunteers involved in this study gave informed consent, and the procedures followed were approved by the institutional ethics committee.
3
Lymphocyte Isolation and RNA Extraction
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Lymphocyte isolation was performed from 5 mL of venous blood, diluted 1:1 with phosphate buffered saline (PBS) (Sigma, St. Louis, MO, USA)/2% fetal bovine serum (FBS) (ThermoFisher Scientific, Waltham, MA, USA). Subsequently, 4 mL of Ficoll-Paque (Gibco ThermoFisher Scientific, Waltham, MA, USA) were added. It was centrifuged at 200× g, and the opaque interface of interest was removed. From 2 × 106 lymphocytes, RNA extraction was performed using an RNeasy Mini kit isolation kit (Qiagen, Hilden, Düsseldorf, Germany), following the manufacturer’s recommendations. RNA extraction was performed just after lymphocyte isolation, aliquoted, and stored at −70 °C until further use. The RNA was quantified, and its integrity was determined from 5 µg in a 1% agarose gel with ethidium bromide and tris-acetate-EDTA buffer visualized in a Kodak Molecular Imaging Software (v.4.5.1) imaging system, while its purity was calculated using the A260/A280 ratio. All the RNA samples used in the present study were considered intact and with an optimal purity value.
4
Lymphocyte Isolation and DNA Extraction
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Lymphocyte isolation was performed from 5 mL of 1:1 diluted whole blood with saline phosphate buffer (PBS) (Sigma, St. Louis, MO, USA)/fetal bovine serum (FBS) at 2% (ThermoFisher Scientific, Waltham, MA, USA); subsequently, 4 mL of Ficoll-paque (Gibco ThermoFisher Scientific, Waltham, MA, USA) was added. The opaque interface of interest was centrifuged and separated; the samples were stored in cryovials with a freezing mixture (Dimethyl sulfoxide (DMSO)) at 5% (Sigma, St. Louis, MO, USA), FBS 20% and a half of Iscove modified Dulbecco (IMDM) (Gibco ThermoFisher Scientific, Waltham, MA, USA) at −80°C until use. Finally, from 2 × 106 lymphocytes, DNA extraction was performed, using the isolation kit (Qiagen, Hilden, Düssseldorf, Germany), following the manufacturer's recommendations. The DNA extraction was performed just before the determination of the TL, and its quality and integrity were subsequently determined.
5
Lymphocyte Isolation and RNA Extraction
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Lymphocyte isolation was performed from 5 mL of venous blood, diluted 1:1 with phosphate buffered saline (PBS) (Sigma, St. Louis, MO, USA)/2% fetal bovine serum (FBS) (ThermoFisher Scientific, Waltham, MA, USA). Subsequently, 4 mL of Ficoll-Paque (Gibco ThermoFisher Scientific, Waltham, MA, USA) were added. It was centrifuged at 200× g, and the opaque interface of interest was removed. From 2 × 106 lymphocytes, RNA extraction was performed using an RNeasy Mini kit isolation kit (Qiagen, Hilden, Düsseldorf, Germany), following the manufacturer’s recommendations. RNA extraction was performed just after lymphocyte isolation, aliquoted, and stored at −70 °C until further use. The RNA was quantified, and its integrity was determined from 5 µg in a 1% agarose gel with ethidium bromide and tris-acetate-EDTA buffer visualized in a Kodak Molecular Imaging Software (v.4.5.1) imaging system, while its purity was calculated using the A260/A280 ratio. All the RNA samples used in the present study were considered intact and with an optimal purity value.
6
Isolation and Culture of Antigen-Specific B Cells
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PBMCs were isolated using Ficoll-Paque and stained with Live/dead Fixable Aqua dead cell stain kit 405 nm (life technologies), CD3-PB (UCHT1, BD), CD14-PB (M5E2, BD), CD19-APC-Cy7 (SJ25C1, BD), CD20-AF700 (2H7, Biolegend) and CD27-PE-Cy7(M-T271, BD). Antigen-specific staining was performed using CCP2-BV605, CCP2-APC and CArgP2-PE, or HC55-APC and HC55-PE [25 (link)] or TT-APC and TT-PE. Antigen-specific single B cell sorts were performed on a BD FACSAria III 4 L cell sorter at the Flow cytometry Core Facility (FCF) of the Leiden University Medical Center (LUMC) in Leiden, Netherlands. The cells were cultured on irradiated CD40L L cells in complex RMPI medium for 10–12 days [26 (link)]. RNA isolation, cDNA production, ARTISAN PCR and sequencing was performed as previously described [27 (link), 28 (link)].
7
Isolation and Cryopreservation of Primary Human T-Cells
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Human primary T-cells were isolated from peripheral blood mononuclear cells (PBMCs), obtained from human buffy coat preparations (Zenbio Inc.), using Ficoll-Paque (Life Technologies Inc., Carlsbad, CA) followed by T-cell isolation using the EasySep Human T-Cell Isolation Kit (Stem Cell Technologies, Seattle, WA). Isolated cells were then cultured in T-cell medium (43% Clicks, 43% RPMI 1640, 2% Glutamax (Life Technologies, Inc., Carlsbad, CA), 10% dFCS (Hyclone Laboratories), 1% non-essential amino acid solution, 1% pen/strep solution, 50 ng/mL IL-7-Fc, 50 ng/mL IL-15-Fc. Both the IL-7 and IL-15 reagents were produced in the Epstein Laboratory and enriched T-cells were frozen for subsequent use in liquid nitrogen.
8
Isolation and Differentiation of Erythroid Cells
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Mononuclear cells were isolated from cord blood (CRB Saint- Louis Hospital, Paris, France) or adult bone marrow donors with written informed consent for research use, in accordance with the Declaration of Helsinki. The ethics evaluation committee of INSERM, the Institutional Review Board (IRB00003888), approved our research project (n. 16-319). Mononucleated cells were separated by Ficoll-Paque (Life Technologies) and purified with a human CD34 MicroBead Kit (cat. n. 130100453, Miltenyi Biotec). A two-step culture method for cell expansion and erythroid differentiation was used to obtain highly stage-enriched erythroblast populations.36 (link),37 (link) Briefly, CD34+ cells (purity 94-98%) were cultured for 7 days with interleukin (IL)6, IL3 (10 ng/mL), and stem cell factor (SCF, 100 ng/mL) to expand hematopoietic progenitors.37 (link) CD36+ erythroid progenitors were purified using magnetic microbeads (CD36 FA6.152 from Beckman Coulter and anti-mouse IgG1 MicroBeads from Miltenyi Biotec). Then, erythropoietin (EPO) was added for 10-18 days to allow erythroid differentiation. For assays of colony-forming cells (CFC, counted at 11-14 days), CD34+ hematopoietic stem cells were seeded in methylcellulose (H4034, StemCell Technologies). TGFbRI inhibitor SB431542 was from Selleckchem (cat. n. S1067)38 (link) and TGFb was from Peprotech (cat. n. 100-21).
9
Isolation and Sorting of TAMs and CD8+ TILs
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The isolation of TAMs and leukocytes was previously described (19 (link)). Briefly, single-cell suspensions were obtained from tumor samples. Leukocytes were further separated from contaminating tumor cells by centrifugation over a 40%–75% Ficoll-Paque (Life Technologies, Thermo Fisher Scientific) gradient at 600g for 30 minutes at room temperature. For sorting TAMs, Ficoll-enriched leukocytes were stained with anti-F4/80, anti-CD11b, anti-Ly6G (1A8, BioLegend), and anti-Ly6C (HK1.4, BioLegend) antibodies and purified by FACS for F4/80+CD11b+Ly6G–Ly6C– cells. For sorting CD8+ TILs, Ficoll-enriched leukocytes were stained with anti-CD4 and anti-CD8 antibodies and purified by FACS sorting for CD8+CD4– cells.
10
Isolation and Stimulation of PBMCs
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PBMCs were isolated by density centrifugation (Ficoll-Paque) 400g x g for 40 min at RT, washed twice in PBS+ 0.5% FBS, and resuspended in RPMI 1640 medium supplemented with L-glutamine (Life Technologies, Carlsbad, California, USA), 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies) to a concentration of 106 cells/mL. Cells were incubated with or without 100 ng/mL lipopolysaccharide (LPS) from E.coli (Sigma-Aldrich, St Louis, MO, USA,) alone or in combination with 10−7 M or 10−8 M dexamethasone (Sigma-Aldrich), for 19 h at 37°C in a humidified atmosphere with 5% of CO2. Cell supernatants and cells (snap frozen in liquid nitrogen) were collected, and stored at -80°C until analysis.
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