All animal experiments were approved by the University Committee on Animal Resources at the University of Rochester and complied with the National Institutes of Health guide for the care and use of Laboratory animals. Bone marrow cells were isolated from 10–14 week old C57BL6/J mice (The Jackson Laboratory, ME, USA) as previously described.(68 (link)) Mice were sacrificed and both femurs and tibias were dissected. Bone marrow was flushed out using MSC culture media (Low Glucose DMEM with 10% FBS, and 1% antibiotic-antimycotic). Cells were filtered through a 70 μm cell strainer (Fisher Scientific), and then collected by centrifugation at 1000 RPM, 4 °C, for 10 min. The collected cells were resuspended in 10 mL MSC culture media (200,00 cells/mL) and plated at cell culture dish (100 × 17 mm, ThermoFisher). When cells achieved about 60% confluence, they were detached using a cell scraper and transferred to 6-well plates (~ 0.4 ×106 cells/cm2) for characterization and differentiation. mMSCs were characterized for putative mesenchymal stem cell markers (positive markers: CD44, CD51 and Sca-1; negative markers: CD45 and CD11b).(69 (link), 70 (link)) Data demonstrated that ~90%, ~86% and ~95% of mMSCs were positive for CD51, Sca-1, and CD44 respectively, ~84% of mMSCs were positive for all the three stem markers, and <8% mMSCs were positive for CD45 and CD11b (Supplemental Fig. 3 ).
Cell culture dish
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The cell culture dish is a basic laboratory equipment used for the in vitro cultivation of cells. It provides a controlled environment for the growth and maintenance of cells.
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4 protocols using cell culture dish
1
Isolation and Characterization of Murine Mesenchymal Stem Cells
2
Fabrication of hUC-MSCs Sheet for Ischemic Heart
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As shown in the flowchart (Fig. 1 ), 5th passage hUC-MSCs were obtained after thawing cells stored in the working cell bank. When cell confluence reached approximately 80%, the hUC-MSCs were collected from the flasks and rinsed with PBS. Then, 6 × 107 hUC-MSCs were suspended in the Cell Sheet Culture Medium (BOE Regenerative Medicine Co. Ltd., Beijing, China), seeded onto a Φ100 mm temperature-responsive cell culture dish (Thermo Fisher Scientific, Waltham, MA, USA), and incubated at 37 °C, 5% (v/v) CO2, and 95% humidity overnight. After that, the culture dish was moved to a biosafety cabinet at room temperature for 40 min. The hUC-MSCs sheet detached from the culture dish spontaneously.![]()
Schematic illustrations of the procedures used for the fabrication of hUC-MSCs sheet and the cytokines secretion from hUC-MSCs sheet to ischemia heart tissue
3
In Vivo Tumor Cell Engraftment Assay
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LAX56 cells were incubated with either idelalisib 5 µM or DMSO as a vehicle control for one hour. The 5 µM concentration used is similar to the Cmax of idelalisib in human trials at tolerated doses [25 (link)]. The cells were washed and injected into the tail vein of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (5 × 106 cells/mouse, n = 6/group). Eighteen hours after injection the mice were euthanized using CO2. Their spleen, bone marrow, blood, and lungs were collected and processed into cell suspensions separately and underwent red cell lysis, then plated in a cell culture dish (ThermoFisher Scientific) coated with methylcellulose-based medium with recombinant human cytokines (Methocult Enriched; Stem Cell Technologies, Vancouver, BC Canada) per the manufacturer’s instructions. 5 × 104 mononuclear cells/dish were plated in triplicate for each organ checked in each mouse. Plates were monitored until sufficient colonies were present to count 20 days later. Colonies were defined as accumulations of >30 cells.
4
Allogeneic T-Cell Cytotoxicity Assay
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Allogeneic T cells from MLPC were used as effectors and the ratio of E:Target (T) was 10:1. Primary patient T cells were used as a source of E and T cells stimulated with no peptide served as a growth control. E and T were incubated together at 37 °C for 4 h, resuspended in IMDM-Medium containing 2% FCS and added to a 3 mL HSC-colony-forming unit complete medium (Miltenyi Biotech, Bergisch Gladbach, Germany), then aspirated using a syringe. A total of 1.1 mL medium was placed into each cell culture dish (Thermo Scientific, Waltham, MA, USA). Colonies were analysed after a 20-day incubation time; the difference between control and sample in percent was calculated and displayed.
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