Case Study: Isolation And Proliferation Of MSC

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2. Materials and Methods:
2.1. Materials:
Natural red, propidium iodide (PI), acridine orange and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St.Louis, MO). May–Grunwald–Giemsa stain was procured from Merck (Darmstadt, Germany). Fetal calf serum, Dulbecco's Modified Eagle Medium (DEMEM) and RPMI 1640 were bought from GIBCO/Life Technologies Inc. (Gaithersburg, MD).
2.2. Isolation and Proliferation of MSCs
Bone marrow derived MSCs were isolated as described previously (Baghaban Eslaminejad et al., 2008). In brief, bone marrow from deep anesthetized wistar rats was flushed out of tibias and femurs. After 2 washings by centrifugation at 1200 rpm for 5 minutes in PBS, cells were plated in 75-cm2 tissue-culture flasks with concentrations of 0.3 to 0.4 × 106 cells/cm2 in the DEMEM medium supplemented with 15% fetal calf serum. Cells were incubated in a humidified 5% CO2 at 37 °C. Four days following primary culture initiation, the culture mediums were collected, centrifuged and the cell
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Incubation of Macrophage with MSCs
100 µl of MSC suspension (2 × 105 cell/ml) was added to each well of 96-well microplates containing macrophages and incubated for 4 h at 37°C in a moist atmosphere of 5% CO2.
2.5 Phagocytic assay by neutral red
The basic phagocytic ability of macrophage (non-opsonic dependent phagocytosis) was measured by neutral red uptake. After MSCs and Macrophages were co-cultured for 2 h, 200 µl neutral red solutions (dissolved in 10 mmol/L PBS with the concentration of 0.075%) was added and incubated for 1h. The supernatant was discarded and the cells in 96-well plates were washed with PBS twice to remove the neutral red that was not phagocytized by macrophages. Then cell lysate (ethanol and 0/01 % acetic acid at the ratio of 1∶1, 200 µl/well) was added to lyse cells. After cells were incubated at 4℃ overnight, the optical density at 490 nm was measured by a microplate reader.
2.6. Phagocytosis Assay by opsonized heat-killed baker's

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