Ten female rats were randomly selected to serve as bone marrow donors for the isolation of marrow-derived c-Kit+ cells. For this purpose, rats were euthanized and the humerus and femur bones were obtained under sterile conditions. The bones were then cut by sterile scissors and marrow contents were flushed with sterile phosphate-buffered saline (PBS) using a 26-gauge needle attached to a 1-mL syringe. Samples were minced and gently triturated to yield a single cell suspension then rinsed in PBS and centrifuged at 400 ×g for 5 minutes. Freshly isolated bone marrow mononuclear cells (MNCs) were obtained using the Ficoll-Paque Plus density gradient medium (Cat no: 17-1440-02; GE Healthcare) according to the manufacturer’s instruction. To enrich c-Kit+ cells using magnetic-activated cell sorting (MACS), MNCs were blocked with 1% BSA for 20 minutes at room temperature, and anti-CD117 microbeads were added to the cell suspension and maintained at 4°C for 1 hour. Cells were then passed through the LS column and both c-Kit positive and negative cells were separated into different conical tubes.
Ficoll paque plus density gradient medium
Manufactured by GE Healthcare
Sourced in United States
Ficoll-Paque PLUS is a density gradient medium used for the separation and isolation of cells and other biological materials. It is a sterile, pyrogen-tested liquid that facilitates the separation of specific cell types or subcellular fractions based on their differences in buoyant density.
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Lab products found in correlation
Ammonium chloride potassium lysing buffer, by Thermo Fisher Scientific (1 mentions)
Thioglycollate, by Merck Group (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
G csf, by Amgen (1 mentions)
Cd34 antibody, by BD (1 mentions)
Amd3100, by Merck Group (1 mentions)
Rhil 2, by R&D Systems (1 mentions)
Anti cd3 antibody, by Miltenyi Biotec (1 mentions)
24 well cell culture plate, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ultra low attachment 96 well plate, by Corning (1 mentions)
9 protocols using ficoll paque plus density gradient medium
1
Isolation of Rat Bone Marrow c-Kit+ Cells
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Ten female rats were randomly selected to serve as bone marrow donors for the isolation of marrow-derived c-Kit+ cells. For this purpose, rats were euthanized and the humerus and femur bones were obtained under sterile conditions. The bones were then cut by sterile scissors and marrow contents were flushed with sterile phosphate-buffered saline (PBS) using a 26-gauge needle attached to a 1-mL syringe. Samples were minced and gently triturated to yield a single cell suspension then rinsed in PBS and centrifuged at 400 ×g for 5 minutes. Freshly isolated bone marrow mononuclear cells (MNCs) were obtained using the Ficoll-Paque Plus density gradient medium (Cat no: 17-1440-02; GE Healthcare) according to the manufacturer’s instruction. To enrich c-Kit+ cells using magnetic-activated cell sorting (MACS), MNCs were blocked with 1% BSA for 20 minutes at room temperature, and anti-CD117 microbeads were added to the cell suspension and maintained at 4°C for 1 hour. Cells were then passed through the LS column and both c-Kit positive and negative cells were separated into different conical tubes.
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Ten female rats were randomly selected to serve as bone marrow donors for the isolation of marrow-derived c-Kit+ cells. For this purpose, rats were euthanized and the humerus and femur bones were obtained under sterile conditions. The bones were then cut by sterile scissors and marrow contents were flushed with sterile phosphate-buffered saline (PBS) using a 26-gauge needle attached to a 1-mL syringe. Samples were minced and gently triturated to yield a single cell suspension then rinsed in PBS and centrifuged at 400 ×g for 5 minutes. Freshly isolated bone marrow mononuclear cells (MNCs) were obtained using the Ficoll-Paque Plus density gradient medium (Cat no: 17-1440-02; GE Healthcare) according to the manufacturer’s instruction. To enrich c-Kit+ cells using magnetic-activated cell sorting (MACS), MNCs were blocked with 1% BSA for 20 minutes at room temperature, and anti-CD117 microbeads were added to the cell suspension and maintained at 4°C for 1 hour. Cells were then passed through the LS column and both c-Kit positive and negative cells were separated into different conical tubes.
2
Tumor Growth Monitoring and Cell Isolation
3
Isolation of Human Mast Cells
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All blood samples were processed within 2 h of collection. Human MCs were isolated from blood using Ficoll-Paque PLUS density gradient medium (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Blood collected in heparinized vacuum tubes was diluted in a 1:1 ratio with sterile phosphate-buffered saline (PBS; pH 7.4; Sigma–Aldrich, St. Louis, MO, USA). This mixture was carefully layered over Ficoll-Paque PLUS medium at a 2:1 ratio before centrifugation at 1700 rpm for 30 min, without brake. The layer containing MCs was carefully aspirated and washed twice in PBS with 0.5% fetal calf serum (FCS; Thermo Fisher Scientific, Waltham, MA, USA) at 1300 rpm for 8 min. MCs were then re-suspended in PBS containing 0.5% FCS. After trypan blue-based counting, MCs were subjected to cell sorting.
4
Isolation of Peritoneal and Splenic Macrophages
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For peritoneal macrophage isolation, mice aged 2–4 months were i.p. injected with 3 mL of 3% thioglycollate (Sigma-Aldrich, T9032). After 3 days, mice were euthanized, and peritoneal cells were recovered by lavage. For isolation of splenic macrophages, mice were euthanized and spleens were dissected and mashed in growth medium (high glucose DMEM with 10% FBS) and passed through a 70 μm strainer. Cells were pelleted and resuspended in red blood cell lysis buffer. Monocytes and lymphocytes were recovered using Ficoll-Paque Plus density gradient medium (GE Healthcare Life Sciences, 17144002) according to the manufacturer’s instructions, and suspension cells (lymphocytes) were washed away prior to experiments.
5
Isolation of Fox PBMCs Using Ficoll-Paque
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Fox PBMCs were isolated on Ficoll-Paque Plus density gradient as described [41 (link),50 (link)]. All heparinized blood was briefly diluted with phosphate-buffered saline containing 2% fetal bovine serum (PBS/2% FBS) in equal quantities. For each blood sample (5–9 mL), 15 mL Ficoll® Paque Plus density gradient medium (GE Healthcare, Waukesha, WI, USA) was filled into a SepMate™-50 centrifugation tube (StemCell Technologies, Inc., Vancouver, BC, Canada) before adding the diluted blood sample on top of the gradient. After centrifugation for 10 min at 1200× g, the PBMC containing fraction was poured off into a clean 50 mL tube and washed 3 times with PBS/2% FBS for 8 min at 300× g. Isolated PBMCs were then suspended in RPMI-1640 medium (Gibco™ [Thermo Fisher Scientific], Waltham, MA, USA) supplemented with FBS (10%), GlutaMAX™-I (2 mM; Gibco™), non-essential amino acids (1%; Gibco™), Penicillin/Streptomycin (100 IU/mL/100 µg/mL; Gibco™), Sodium Pyruvate (1 mM; Gibco™), Gentamicin (20 µg/mL; Gibco™) and 2-Mercaptoethanol (50 µM; Gibco™).
6
Isolation and Purification of Rhesus Macaque HSPCs
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Rhesus macaque PBMCs were isolated from PB using Ficoll-Paque PLUS density gradient medium (GE Healthcare) following manufacturer recommendations. G-CSF-mobilized (Amgen) and plerixafor-mobilized (Amgen) rhesus macaque CD34+ HSPCs were collected as described previously.29 (link),60 (link) In short, the animals were treated with a 5-day course of 15 μg/kg G-CSF (Amgen) subcutaneously and a single subcutaneous dose of 1 mg/kg AMD3100 (Sigma-Aldrich) on the morning of the fifth day, 3–4 h before leukapheresis. A small-volume leukapheresis procedure was performed using a CS3000 cell separator (Baxter Fenwal), and CD34+ HSPCs were immunoselected using a rhesus macaque CD34+ antibody (clone 12.8, Fred Hutchinson Cancer Research Center) and an anti-mouse IgM bead (Miltenyi Biotech). A CD34+ antibody (550761, BD Biosciences) was used to determine the purity of the immunoselected population.
7
Expansion of Cytokine-Induced Killer Cells
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After approval by the institutional review board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA# 774/2017) and written informed consent from a healthy donor were obtained, PBMCs were isolated from the peripheral blood of the healthy donor (n = 3) by standard Ficoll separation using Ficoll-Paque Plus density gradient medium (GE Healthcare). Then, the PBMCs were plated in a 24-well cell culture plate (Thermo Fisher) at 1 × 10 6 cells per well in 2 ml (0.5 × 10 6 cells/ml) of culture medium consisting of RPMI-1640 medium (Hyclone) containing 10% FBS (Gibco) with 1,000 IU/ml recombinant human IFN-γ (rhIFN-γ) (Miltenyi) and incubated at 37°C in a humidified atmosphere containing 5% CO 2 . After 24 h of incubation, 50 ng/ml anti-CD3 antibody (Miltenyi) and 500 IU/ml rhIL-2 (R&D Systems) were added to the culture medium. On days 4, 7, and 11 of culture, RPMI 1640 medium containing 10% FBS and 500 IU/ml rhIL-2 was added to maintain the cell density at 1 × 10 6 cells/ml, and the cells were plated in a 24-well cell culture plate at a maximum density of 2 ml/well. On day 14 of culture, CIK cells were harvested for further analysis.
8
Isolation of Mesenchymal Progenitor Cells
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Mesenchymal progenitor cells were allowed to attach to T175 cell culture flasks (Cellstar, Greiner Bio-One GmbH) for 24–48 h. Non-adherent cells were then collected and mononuclear cells were further purified using the Ficoll-Paque PLUS density gradient medium (GE Healthcare). Each sample was diluted with an equal volume of warm (+ 35 °C) Ca2+- and Mg2+-free phosphate-buffered saline (PBS, Sigma–Aldrich). The diluted sample was carefully layered on top of Ficoll-Paque solution and centrifuged at 500g for 30 min at room temperature. The monocyte fraction was collected, resuspended in warm PBS and centrifuged at 100g for 10 min at room temperature. Finally, the cells were counted in a hemocytometer and used immediately or frozen and stored in liquid nitrogen.
9
Evaluating GBM Cell Sphere Interactions
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PBMCs were obtained from healthy human donors as approved by the Institutional Review Board (IRB) at Brigham and Women’s Hospital (all samples were de-identified) and isolated with Ficoll-Paque PLUS density gradient medium (GE Healthcare Life Sciences) according to the manufacturer’s instructions. A single-cell suspension of GBM cells was seeded at 2,000 cells/well (G9pCDH) in ultra-low-attachment 96-well plates (Corning) and incubated for 24 h to allow sphere formation. PBMCs were then added with the different ADU-S100 concentrations, and treatment was repeated 3 d later. Cells were then incubated for another 3 d (total = 6 d of coculture). Microscope images of the spheres were taken daily (Nikon TI, 4× magnification), and the spheres’ fluorescence was measured in ImageJ with a dedicated macro.
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