Primary rest NK cells were separated from peripheral blood mononuclear cells (50mL; from healthy donors). The mononuclear cells were purified using Ficoll Paque density gradient centrifugation with 15mL falcons (BD, CA). Cell sorting from peripheral blood mononuclear cell was performed using the antibody-conjugated magnetic beads (Miltenyi Biotech, Gladbach, Germany) by negative selection kits, according to the manufacturer’s instructions. Twenty million mononuclear cells were used as the input count of cells for all the bead sorting. Mononuclear cells underwent 2 rounds of sorting through CD3 and thereafter CD14 beads (2mL beads/1 million cells). Flow cytometry was used to assess enrichment populations (more than 95% purity). The process was followed by expansion and activation in XVIVO-20 medium (Lonza, Barcelona, Spain) supplemented with 10% fetal bovine serum (Gibco, UK), 500IU/mL IL-2 (Promokine, Germany), 10ng/mL IL-12 (Promokine), 100ng/mL Galactosyl ceramide, 50ng/mL IL-15 (Promokine), 1μM Valproic acid, and 10ng/mL IL-21 (Promokine) for about 5 days.
Ficoll paque
Manufactured by BD
Sourced in United States
Ficoll-Paque is a sterile, density gradient medium used for the isolation and purification of cells, particularly mononuclear cells, from whole blood or bone marrow. It facilitates the separation of different cell types based on their differences in density.
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Lab products found in correlation
Fibronectin coated dishes, by BD (2 mentions)
Cfu hill medium, by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Antibody conjugated magnetic beads, by Miltenyi Biotec (1 mentions)
X vivo 20 medium, by Lonza (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
Cd4 cf594, by BD (1 mentions)
Anti human cd45 fitc, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
8 protocols using ficoll paque
1
Isolation and Expansion of Primary rNK Cells
2
Quantifying Early Endothelial Progenitor Cells
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From 45 mL of intravenous blood, measures of EPC-CFU were obtained by isolating early EPCs using Ficoll Paque and seeding 5 million cells on 6-well Fibronectin-coated dishes (BD Biosciences) in CFU-Hill medium (Stem Cell Technologies, cat#05900). Non-adherent cells were collected 48 hours later, and 1 million cells seeded on 24 well Fibronectin-coated dishes. On day 5, EPC-CFUs were counted in 5 wells. The mean EPC-CFU was used in analyses as previously described [14 (link), 39 (link)].
3
Immunomodulatory Effects of LPS-Primed AS-MSCs
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AS-MSCs were primed with 1 µg/mL LPS for 4 hours prior to co-culturing with PBMCs to activate TLR4. PBMCs were harvested from blood samples collected from HDs using the Ficoll-Paque density gradient centrifugation method and then labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; BD Bioscience, USA). PBMCs were incubated with 5 µM CFSE in phosphate-buffered saline (PBS) at 37 °C for 5 mins. Following incubation, the staining reaction was terminated by washing the PBMCs with complete medium twice. For co-culturing with AS-MSCs (effector cells), AS-MSCs at a density of 1×105 cells/well seeded in six-well plates were stimulated with or without 1 µg/mL LPS for 4 hours and then subjected to Co-60 irradiation (30 Gy). CFSE-labeled PBMCs (responder cells) at a cell density of 1×106 were then added to the MSC cultures. The co-cultures were stimulated with human anti-CD3/CD28 monoclonal antibodies (mAbs; CD3: 200 ng/mL; CD28: 1 µg/mL, BD Bioscience, USA) for 5 days, after which the PBMCs were harvested and stained with an anti-CD4-FITC antibody (BD Bioscience, USA). Cell proliferation was evaluated using flow cytometry. A minimum of 10,000 live cell events gated in scatter plots were analyzed for each sample.
4
Immune Cell Isolation and Sorting
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Whole blood was collected from each subject into four 10‐ml EDTA collection tubes. PBMCs were isolated using a Ficoll‐Paque density gradient and stained with conjugated monoclonal antibodies against CD45 (fluorescein isothiocyanate conjugated), CD19 (phycoerythrin [PE] conjugated), CD45RA (PE–Cy7 conjugated) (all from BD PharMingen), CD3 (Brilliant Violet 421 conjugated), CD4 (CF594 conjugated) (both from BD Horizon), CD14 (allophycocyanin [APC] conjugated) (BD Biosciences), and CD27 (APC–eFlour 780 conjugated) (ebioscience). Cells were then stored overnight in buffer at 4°C and sorted the following day, on a BD Biosciences FACSAria fluorescence‐activated cell sorter (FACS). The following populations were gated for sorting following exclusion of debris and doublets: monocytes (CD45+CD14+), B cells (CD45+CD14 −CD3 −CD19+), naive CD4+ T cells (CD45+CD14 −CD19 −CD3+CD4+CD27+CD45RA+), and memory CD4+ T cells (CD45+CD14 −CD19 −CD3+CD4+CD45RA−). Cell counts and purity checks were performed after sorting, and then cells were stored frozen as a pellet at −80°C.
5
Isolation and Quantification of EPCs
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Peripheral blood samples were obtained from patients before and three months after MSC injection. EPCs were isolated from samples using Ficoll-Paque and five million cells were seeded on 6-well fibronectin-coated dishes (BD biosciences) in CFU-Hill medium (stem cell technologies, cat#05900) (Hill et al., 2003 (link), Solomon et al., 2012 (link)). The non-adherent cells were collected 48 h later and one million cells were seeded on 24-well fibronectin-coated dishes. On day five, EPC-CFUs were counted in five wells and the average was obtained.
6
Isolation and Characterization of Endothelial Progenitor Cells
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A 15-ml blood sample for the EPCs will be collected in a Sodium Heparin Vacutainer™ tube. Peripheral blood mononuclear cells (PBMC) will be isolated using Ficoll-Paque (GE Heathcare Life Sciences, Uppsala, Sweden). Progenitor cells (PCs) will be defined as CD34+/CD45dim, and EPCs as CD34+/KDR+/CD45dim. Briefly, blood samples will be diluted once with phosphate buffered saline (PBS) and layered onto Ficoll-Paque in 15-ml conical tubes (BD Biosciences, San Jose, CA, USA). Each tube will be centrifuged at 400 xg for 30 min (Eppendorf-5810, Hamburg, Germany), and the PBMCs at the interface will be collected. Cells will be washed once with RPMI-1640 medium (Gibco™, Vancouver, Canada); stained with 5 μl of antihuman-CD45 FITC, 5 μl of antihuman-CD34 PE (clone 6G12), and 8 ul of antihuman-KDR-Alexa Fluor 647 (BD Biosciences, San Jose CA, USA); and incubated in the dark for 30 min, followed by the addition of 500 μl of PBS until resuspension and acquisition in a flow cytometer (FACSCalibur, CellQuest software, BD Biosciences, San Diego, CA, USA). The total number of events recorded on the mononuclear cells gate was 200,000. The percentage of CD34+ cells was calculated based on the measured number of leukocytes (CD45+ cells) using the ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy [43 ].
7
Isolation of Peripheral Blood Mononuclear Cells
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Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from full blood using Ficoll-Paque (GE Health Care, Chicago, IL) centrifugation. Ficoll-Paque (10 mL) was placed at the bottom of a 50 mL Falcon tube and blood was slowly layered above. After being centrifuged (600 g for 30 min at 18°C, no brakes), a layer of PBMCs would be visible and collected for further experiments.
8
Isolation of Peripheral Blood Mononuclear Cells
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Samples of peripheral venous blood were collected from subjects in anticoagulant (heparin 15,000 IU/5 ml; Biochemie GmbH, Kundl, Austria). The peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood by centrifugation on a Ficoll-Paque™ Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient. The blood samples were transferred to 16 ml Falcon (Becton-Dickinson, Brea, CA, USA) sterile tubes and diluted at 1:1 with PBS. Each sample was transferred to a 50 ml Falcon sterile tube with Ficoll-Paque, so that the ratio of the diluted sample: Ficoll was 1:1. The transfer of diluted biological samples over the Ficoll layer was gentle, without mixing the two components, allowing the formation of a density gradient. The samples were centrifuged at 400 × g for 30 min. After centrifugation, the ring of mononuclear cells at the interface of the components was harvested and moved to a sterile tube. This step was followed by two successive washes with PBS and consecutive centrifugations at 100 × g for 10 min. After the second centrifugation, the supernatant was removed, and the cell pellet was used for further analysis, or frozen at −80°C (or −196°C) and kept for further investigation.
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