The sphere-forming assay was performed as described previously [20 (link)]. Cells were cultured in serum-free DMEM/F12 medium (Invitrogen, USA) supplemented with 4 μg/ml heparin (Sigma, USA), 2% B27 supplement (1:50 dilution; GIBCO, USA), 20 ng/ml human recombinant epidermal growth factor (PeproTech, USA), 20 ng/ml human recombinant basic fibroblast growth factor (bFGF) (PeproTech, USA), 100 IU/ml penicillin and 100 μg/ml streptomycin, in an Ultra Low Attachment Culture Flask (Corning, USA). After culturing for 1 week, the number of spheres was counted under a microscope (Leica, Germany).
Ultra low attachment culture flask
Manufactured by Corning
Sourced in United States
Ultra-low attachment culture flasks are designed for the culture of cells that do not readily adhere to standard tissue culture surfaces. They feature a hydrophilic, non-toxic surface that prevents cell attachment, promoting the formation of spheroids, cell aggregates, and suspension cultures.
Automatically generated - may contain errors
Lab products found in correlation
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
Heparin, by Merck Group (1 mentions)
Recombinant human basic fibroblast growth factor, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Cd14 magnetic microbeads, by Miltenyi Biotec (1 mentions)
Recombinant human il 4, by R&D Systems (1 mentions)
Recombinant human gm csf, by R&D Systems (1 mentions)
Quadromacs system, by Miltenyi Biotec (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
5 protocols using ultra low attachment culture flask
1
Sphere-forming Assay for Stem Cells
2
Adipose-Derived Stem Cell Isolation and Expansion
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Adipose tissue was collected from healthy individuals, following approval of an informed consensus. Lipoaspirate samples were harvested from different body areas such as abdomen, breast, flanks, trochanteric region. After mechanic (shake 30 minutes at 37°C) and enzymatic (collagenase 150 mg/ml, Gibco, Carlsbad, CA) digestion, the samples were centrifuged at 1200 rpm for 5 minutes and the stromal vascular fraction (SVF) was resuspended in specific medium. For 3D cultures, the SASCs were plated in stem cell medium (SCM) composed J o u r n a l P r e -p r o o f principally by DMEM/F12 salts with added basic fibroblast growth factor (bFGF, 10 ng/ml; Sigma, St. Louis, MO) and epidermal growth factor (EGF, 20 ng/ml, Sigma) [43] (link). SASCs were grown as fluctuating spheroids in an ultra-low attachment culture flask (Corning, NY) [5] (link). While for 2D cultures, the SVF was plated in DMEM and FBS (10%) in adherent cell culture conditions. All systems were added of a small volume of Antibiotic-Antimycotic solution (1X, Gibco) to prevent bacterial and fungal contamination. Culture flasks were placed at 37°C in a humidified 5% CO2 incubator.
3
Monocyte-derived Dendritic Cell Generation
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CD14+ mononuclear cells were purified from PBMCs via CD14 magnetic microbeads (Miltenyi Biotec, Bergisch, Gladbach, Germany; Cat: #130-050-201) using the QuadroMACS system (Miltenyi Biotec, Bergisch, Gladbach, Germany; Cat: #130-090-976) according to the manufacturer’s recommendations. Cells were cultured at a density of 0.3 × 106 cells/mL in warm CellGenix DC medium (Sartorius CellGenix GmbH, Freiburg, Germany; Cat: #20801-0500) supplemented with 1% (v/v) Glutamax (Gibco Thermo Fisher Scientific Inc., Waltham, MA, USA; Cat: #35050-061), 1% (v/v) non-essential amino acids (Gibco; Cat: #11140-035, 1% (v/v) sodium pyruvate (Gibco Thermo Fisher Scientific Inc., Waltham, MA, USA; Cat: #11360-039), 1% (v/v) penicillin–streptomycin (Gibco Thermo Fisher Scientific Inc., Waltham, MA, USA; Cat: #15140-122). CD14+ cells were plated at 2.5 × 106 cells per sample in 25 cm2 ultra-low attachment culture flasks (Corning Inc., Corning, NY, USA; Cat: #4616), and differentiated into immature moDCs using 5 ng/mL recombinant human IL-4 (R&D Systems, Minneapolis, MN, USA; Cat: #204-IL) and 50 ng/mL recombinant human GM-CSF (R&D Systems, Minneapolis, MN, USA; Cat: #215GM-500) for 5 days at 37 °C in an atmosphere of 5% CO2.
4
Culturing and Expanding Sphere Cells
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Sphere cells were cultured as described previously but with the following modifications [10 (link), 34 (link), 35 (link)]. Monolayer cells were harvested by trypsinization when the flasks were approximately 75% confluent and cultured in DMEM/F-12 supplemented with 0.4% bovine serum albumin or BSA, 10 ng/mL b-FGF, 20 ng/mL EGF, and 5 μg/mL insulin. Cells were cultured as single-cell suspensions in ultralow attachment culture flasks (Corning Inc., Corning, NY) for 10 to 14 days. Spheres were collected every 3 – 4 days by gravity to remove dead and non-sphere forming cells in the supernatant. The spheres were washed with PBS, and separated into single cell suspensions by incubating with 1 mL of 0.25% trypsin-EDTA for 2 – 4 min at room temperature. To generate sphere-derived cells (SDCs) for short-term proliferative growth studies, single cell suspensions of sphere cells were cultured on adherent tissue-culture treated flasks in complete medium supplemented with 75 μg/mL Matrigel™ Basement Membrane Matrix Growth Factor Reduced (Stemcell Technologies Inc., Vancouver, BC, Canada).
5
Megakaryocyte Maturation and Proplatelet Formation
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Unless stated otherwise, cells were cultured in nontissue culture-treated flasks (BD Biosciences, Franklin Lakes, NJ, USA).
Proplatelet formation was induced in DM. For maturation of MEG-01 cells, we used ultra-low attachment culture flasks (Corning, Corning, NY, USA). Before transferring cells to DM, the cells were centrifuged at 200 g for 5 min to separate megakaryocytes from spontaneously matured cells. To pretreat cells with an ER stressor, megakaryocytes were suspended in GM containing TG. These pre-treated cells were collected by centrifugation at 200 g for 5 min and then resuspended in DM. Cells were isolated from cultures after 24 and 48 h.
To isolate mature megakaryocytes from DM cultures, megakaryocytes were first separated by centrifugation at 200 g for 5 min, and then the supernatant was centrifuged at 1500 g for 15 min to recover proplatelets and PLPs. Isolated megakaryocytes and mature cells were suspended in PBS for flow cytometric analysis and light microscopy, or in sample buffer for Western blot analysis (see below).
Proplatelet formation was induced in DM. For maturation of MEG-01 cells, we used ultra-low attachment culture flasks (Corning, Corning, NY, USA). Before transferring cells to DM, the cells were centrifuged at 200 g for 5 min to separate megakaryocytes from spontaneously matured cells. To pretreat cells with an ER stressor, megakaryocytes were suspended in GM containing TG. These pre-treated cells were collected by centrifugation at 200 g for 5 min and then resuspended in DM. Cells were isolated from cultures after 24 and 48 h.
To isolate mature megakaryocytes from DM cultures, megakaryocytes were first separated by centrifugation at 200 g for 5 min, and then the supernatant was centrifuged at 1500 g for 15 min to recover proplatelets and PLPs. Isolated megakaryocytes and mature cells were suspended in PBS for flow cytometric analysis and light microscopy, or in sample buffer for Western blot analysis (see below).
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