Nt-SCs were isolated from fresh bone marrow samples derived from the iliac crest under the approval of the Ethics Committee of the University of Heidelberg. Bone marrow cells were purified on a Ficoll-Paque™ Plus density gradient (GE Healthcare, München, Germany), washed in PBS and treated with erythrocyte lysis buffer (0.154 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) to remove erythrocytes. The nt-SC-enriched fraction was seeded and cultured in DMEM high-glucose supplemented with 12.5% FCS (Biochrom), 2 mM L-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (Lonza), 4 ng/ml basic fibroblast growth factor (Merck Chemicals GmbH, Darmstadt, Germany, 50 μM 2−mercaptoethanol and 1% non-essential amino acids (Invitrogen, Karlsruhe, Germany). After 48 h, cultures were washed with PBS to remove non-adherent material. During expansion, medium was replaced twice a week.
Ficoll paque plus density gradient
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom, United States, Germany
Ficoll-Paque Plus is a density gradient medium used for the separation and purification of cells, organelles, and other biological particles. It is a sterile, pyrogen-free solution that creates a density gradient upon centrifugation, allowing the separation of different cell types based on their density.
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Lab products found in correlation
Penicillin streptomycin, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Human serum, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Il 15, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Cd4 positive isolation kit, by Miltenyi Biotec (1 mentions)
M1882, by Merck Group (1 mentions)
Iscove s modified dulbecco s medium imdm, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Cd3 microbeads, by Miltenyi Biotec (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
40 protocols using ficoll paque plus density gradient
1
Isolation and Expansion of Nucleated Stem Cells
2
CD4+ T Cell Isolation from OC, BOT, and HC
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Fresh peripheral blood was collected from 20 patients with OC, 15 patients with BOT, and 15 age-matched HC. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare Bio-Sciences, Sweden). CD4+ T cells were then separated by CD4 positive isolation kit (Miltenyi Biotec, Germany). Isolated CD4+ T cells purity was higher than 95% as determined by flow cytometry.
3
Profiling PBMC response to inflammatory stimuli
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Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coats of blood samples from healthy volunteers by centrifugation through a Ficoll-Paque PLUS density gradient (1,077 g/ml, GE Healthcare, Chicago, Illinois, USA). PBMC were washed thrice with PBS and 7.106 cells were grown for 2 h in M-SFM medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Monocytes were separated from PBMC by elimination of suspended cells. Then, PBMC-derived monocytes were cultured for 24 h with myoglobin (250 µM, M1882; Sigma-Aldrich, Saint-Quentin Fallavier, France), ibrutinib (3 µM, Imbruvica caps, Janssen Pharmaceutica, Belgium), uromodulin (6.25 mg/ml, Origen Biomedical, Burleson, Austin, USA), or medium alone.
Regarding the human blood samples, all the patients gave written informed consent and the samples collections was approved by ethical committee of the French Administration of Blood Products (Etablissement Français du Sang, authorization number 21PLER2017-0016). The study was performed according to the French law regarding human studies, and fulfilled the recommendations of the Helsinki’ conference, as revised in 2004.
Regarding the human blood samples, all the patients gave written informed consent and the samples collections was approved by ethical committee of the French Administration of Blood Products (Etablissement Français du Sang, authorization number 21PLER2017-0016). The study was performed according to the French law regarding human studies, and fulfilled the recommendations of the Helsinki’ conference, as revised in 2004.
4
Isolation and Culture of NANT Cells
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NANT cells were isolated as previously described [8 ]. Briefly, PBMCs were isolated from peripheral blood using Ficoll-Paque™ PLUS density gradient centrifugation (GE HealthCare, Uppsala, Sweden). Adherent cells were depleted by adherence, and T cells in the non-adherent fraction of PBMCs were removed using CD3+ MicroBeads according to the manufacturer’s instructions (Miltenyi Biotec, California, USA). The NANT cells were then incubated in Iscove’s Modified Dulbecco’s Medium (IMDM; Sigma-Aldrich) supplemented with 30% FBS (Sigma-Aldrich) and 1% BSA in a humidified incubator, at 37∘C with 5% CO2, for 3 days in the presence or absence of treatments. NANT cells were counted on a haemocytometer after Trypan blue staining.
5
Whole Blood, PBMC, and Plasma Preparation
6
Isolation of Mononuclear Leukocytes from Cord Blood
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At the time of delivery, the umbilical cord was disinfected and cut at the placental side of the clamp. Cord blood was collected in sterile EDTA tubes, mixed thoroughly, and checked for blood clots. Mononuclear leukocytes were isolated by Ficoll-Paque Plus density gradient (GE Healthcare Biosciences, Pittsburgh, PA), following the manufacturer’s protocol and the method of Normann et al.55 (link). In brief, collected blood was diluted with an equal amount of phosphate buffered saline (PBS). 1 ml of PBS was mixed thoroughly with 16 ml of Ficoll-Paque plus and used as density gradient. 25 to 30 ml of diluted blood was layered on top of the Ficoll mixture and centrifuged at 400xg for 30 minutes. The upper plasma layer was collected and centrifuged to remove platelets. The cellular interface was then collected in a separate tube and washed with PBS containing 1 mM EDTA. Cells were washed with autologous plasma to remove platelets. Finally, the mononuclear leukocytes were washed with PBS, and 2 million packed cells per vial were saved at −80 °C for RNA isolation and microarray analysis.
7
Isolation of Naive Human B Cells
8
Serum and PBMC Isolation Protocol
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Serum was collected from whole blood in 5-ml tubes without anticoagulant, centrifugated at 2500 rpm for 15 min, aliquoted, and stored at −20°C for further experiments. PBMCs were isolated from blood collected in K3EDTA tubes by density gradient centrifugation. Briefly, PBMCs were isolated by Ficoll-paque plus density gradient (GE) centrifugation at 2000 rpm for 20 min at room temperature. PBMCs were washed 2 times in phosphate-buffered saline and subsequently frozen in liquid nitrogen in 90% fetal bovine serum (FBS, Gibco) with 10% DMSO (Gibco) for further immune profile analysis.
9
Isolation and Characterization of Immune Cells
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PBMCs and SFMCs were extracted using Ficoll‐Paque PLUS density gradient centrifugation (GE Healthcare Life Sciences, Chicago, IL, USA), and stored frozen in FCS+10% DMSO. Jurkat and JPRM441 (low RasGRP1) cells were previously characterized and described [25 , 37 ]. RPMI1640 containing 10 mM HEPES, 2 mM l ‐glutamine, 100 U/mL Penicillin–streptomycin, and 10% FCS was used as culture medium. Starvation was performed in plain RPMI1640 for 30 min. When indicated cells were stimulated with PMA (Sigma‐Aldrich, Zwijndrecht, the Netherlands, 20 ng/mL), ionomycin (Sigma‐Aldrich, 1 μM).
10
Isolation and Cryopreservation of Human PBMCs
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Human PBMCs were isolated from buffy coats of healthy volunteers (obtained at Portuguese Institute of Blood and Transplant) by a Ficoll-Paque PLUS density gradient (GE Healthcare, UK). PBMCs were then resuspended in RPMI-1640 medium (Lonza, Switzerland), supplemented with 10% (v/v) heat-inactivated FBS (HyClone, USA), 2 mM of L-glutamine (Lonza, Switzerland), 1% (v/v) Pen/Strep/Fungiezone solution (HyClone, USA) and placed at 37 °C in a humidified atmosphere of 5% CO2 overnight. After this recovering step, PBMCs were counted and frozen in freezing medium (10% DMSO, v/v, in FBS) and stored at −80 °C. When needed, PBMCs were thawed and cultured in the same supplemented-RPMI-1640 medium as before. PBCMs were maintained overnight at 37 °C in a humidified atmosphere of 5% CO2, to decrease loss of effector capacity65 (link) and only then used in the ADCC assays.
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