Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood samples using the Ficoll density gradient separation (GE Healthcare, Chicago, IL, USA) according to the manufacturer's protocols. A portion of the PBMCs was subjected to T cell separation using anti-human CD3 beads (STEMCELL Technologies, Vancouver, Canada); isolated cells were measured using flow cytometry. To differentiate human naïve CD4+ T cells into Th1 cells, naive CD4+ T cells were separated using an EasySep human naïve CD4+ T cell isolation kit (STEMCELL Technologies) to negatively select for CD4+CD45RO− cells from PBMCs according to the manufacturer's protocols. The naïve CD4+ T cells were stimulated with human CD3/CD28 T cell activator (STEMCELL Technologies) and human Th1 differentiation supplement (STEMCELL Technologies), and were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. On day 3, differentiated Th1 cells are ready for use.
Ficoll density gradient separation
Manufactured by GE Healthcare
Sourced in United States
Ficoll density gradient separation is a centrifugation technique used to isolate specific cell types or subcellular components from a complex mixture. It utilizes a non-ionic, hydrophilic polysaccharide polymer to create a density gradient, allowing the separation of different cell populations or organelles based on their density during centrifugation.
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Lab products found in correlation
Human cd3 cd28 t cell activator, by STEMCELL (1 mentions)
Easysep human na ve cd4 t cell isolation kit, by STEMCELL (1 mentions)
Facsaria 2, by BD (1 mentions)
Facs buffer, by Thermo Fisher Scientific (1 mentions)
Pha l, by Merck Group (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Nontissue culture treated dishes, by Thermo Fisher Scientific (1 mentions)
M csf, by R&D Systems (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
Human bm mncs, by Lonza (1 mentions)
Aim 5, by Thermo Fisher Scientific (1 mentions)
Human serum albumin (hsa), by CSL Behring (1 mentions)
Cryostor cs10 cell freezing medium, by STEMCELL (1 mentions)
Anti cd43 magnetic beads, by Miltenyi Biotec (1 mentions)
Iscove s modified dulbecco s medium, by Lonza (1 mentions)
7 protocols using ficoll density gradient separation
1
Isolating Naïve CD4+ T Cells and Inducing Th1 Differentiation
2
Isolation and FACS Sorting of CD34+ BMMCs
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BMMCs were isolated from whole bone marrow aspirate by Ficoll density gradient separation (GE Healthcare), resuspended in 90% FBS + 10% DMSO, and cryopreserved in liquid nitrogen. To prepare cells for FACS, frozen BMMCs vials were thawed in a 37 °C water bath for 2 min. Vials were removed once only a tiny ice crystal was left. Thawed BMMCs were washed and resuspended in 1 PBS and 20% FBS. After recovery, FACS sorting was performed on a Becton Dickinson FACSAria II (BD Biosciences, Denmark) to remove the dead cells. BMMCs were stained with pre-conjugated CD34-PE antibody for 15 min on ice. Non-specific binding was blocked by incubation in FACS buffer (Life Technologies). The unbound antibodies were removed using 5 ml wash buffer. The CD34+ cells were sorted out by FACS Aria™ II. The final concentration of thawed cells was 1 x 106 cells per ml.
3
Isolation and Activation of Human CD4+ T Cells
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Human blood samples were obtained from healthy adult donors following informed consent in accordance with the Declaration of Helsinki under a research protocol approved by the Research Ethics Review Board of the Institut de Recherches Cliniques de Montréal (IRCM). Peripheral blood mononuclear cells (PBMCs) were purified from buffy coats following Ficoll density gradient separation (GE Healthcare). CD4+ T cells were isolated by negative selection using a CD4+ T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. Purified CD4+ T cells were activated with 5 μg/ml phytohemagglutinin-L (PHA-L; Sigma-Aldrich, 11249738001) and 100 U/ml interleukin-2 (IL-2) (PeproTech, 200-02) for 3 days and cultured in RPMI-10 containing 100 U/ml IL-2 for another 2 days before infection.
PBMCs were seeded for 2 h at 37°C in nontissue culture-treated dishes (Fisherbrand) containing serum-free RPMI medium. After gentle washes, adherent cells (which mostly contain monocytes) were cultured for 7 days in RPMI supplemented with 5% decomplemented autologous human blood plasma and 10 ng/ml macrophage colony-stimulating factor (M-CSF) (R&D Systems, 216-MC) to obtain MDMs. Purity of MDMs was determined by CD11b surface staining and was found to routinely reach greater than 95%.
PBMCs were seeded for 2 h at 37°C in nontissue culture-treated dishes (Fisherbrand) containing serum-free RPMI medium. After gentle washes, adherent cells (which mostly contain monocytes) were cultured for 7 days in RPMI supplemented with 5% decomplemented autologous human blood plasma and 10 ng/ml macrophage colony-stimulating factor (M-CSF) (R&D Systems, 216-MC) to obtain MDMs. Purity of MDMs was determined by CD11b surface staining and was found to routinely reach greater than 95%.
4
Nasal Biopsy and Gene Expression
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Nasal biopsy specimens (2.5 mm) were taken from the inferior turbinate after achievement of local anesthesia and subsequently homogenized with a Qiagen TissueLyser (Qiagen, Hilden, Germany). Peripheral blood lymphocytes were isolated from venous blood by using Ficoll density gradient separation (GE Healthcare, Fairfield, Conn). Total RNA was extracted with the RNeasy Mini Kit (Qiagen), and cDNA was synthesized by using SuperScript III RT (Invitrogen, Carlsbad, Calif).
5
Isolation and Cryopreservation of Human Bone Marrow-Derived Mononuclear Cells
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Human BM-MNCs were purchased from Lonza (Basel, Switzerland). The MNCs were extracted from the whole bone marrow of healthy donors via Ficoll density gradient separation (GE Healthcare, ORD, USA), which was then frozen. In our study, we used five lots. Donors included HIV/HBV/HCV-negative women and men with an age range of 22–35.
6
Expansion and Cryopreservation of T Cells
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PBMCs were isolated from leukapheresis units using Ficoll density gradient separation (GE Healthcare, Chicago, IL, USA) and cryopreserved. PBMCs were thawed and cultured in AIM-V (Thermo Fisher Scientific, Waltham, MA) supplemented with 5% CTSTM Immune Cell Serum Replacement (SR) (Thermo Fisher Scientific, Waltham, MA) and human IL-2 (300 IU/ml, Quangang, Shandong, China). PBMCs were activated and expanded with CTSTM DynabeadsTM CD3/CD28 (Thermo Fisher Scientific, Waltham, MA) for 24 h. T cells were transduced with LVs at multiplicity of infection (MOI) of 1. Three days after transduction, the cells were extensively washed to remove the LV particles. T cells were further expanded in AIM-V medium supplemented with 5% CTSTM Immune Cell SR (Thermo Fisher Scientific, Waltham, MA) for three to ten days in the presence of IL-2 (300 IU/ml, Quangang). The cell product was washed twice with normal saline (Qidu pharmaceuticals, Shandong, China) containing 2.5 % human serum albumin (CSL Behring, King of Prussia, PA, USA). Cells were resuspended in Cryostor® CS10 Cell Freezing Medium (STEMCELL Technologies, Vancouver, Canada) for cryopreservation and stored in liquid nitrogen.
7
Mesenchymal Stem Cells Modulate B Cell Activation
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Splenocytes were isolated from spleens of deceased organ donors (Biobank Erasmus MC protocol No. MEC-2012-022) by Ficoll density gradient separation (GE Healthcare, Uppsala, Sweden). Quiescent B cells were obtained by negative selection using anti-CD43 magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). B cells were cultured for 7 days in Iscove's modified Dulbecco's medium (Lonza) supplemented with 10% HI FBS and stimulated with F(ab)2 anti-IgM ( Jackson, ImmunoResearch laboratories, Inc., West Grove. PA), IL-2 (10 3 IU, Proleukin; Prometheus Laboratories, Inc., San Diego, CA), and 5 mg/mL anti-CD40 agonistic monoclonal antibody (Bioceros, Utrecht, The Netherlands). Inactivated and control MSC were added to the culture at day 0 in a MSC:B cell ratio of 1:5. IL-10 levels in the supernatant were measured using a human IL-10 ELISA kit (U-Cytech, Utrecht, The Netherlands) according to the manufacturer's protocol.
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