Impact of Oxygen-Glucose Deprivation and Reoxygenation on Cell-Cell Interactions

プロトコル

1. Seeding of Endothelial Cells

1. Obtain primary cultures of RBMECs from adult Sprague Dawley rats (or obtain them commercially). 

2. Cultivate RBMECs in 100 cm fibronectin (50 µg/ml) coated petri dishes using the rat brain endothelial cell growth medium. Change the medium every two days until confluency is reached. 

3. On reaching 80-90% confluency, gently wash the cells in 5 ml phosphate buffered saline (PBS) by swirling. The cells are then detached by exposing them to 1 ml of warm 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA) solution, equilibrated to 37 °C. 

4. Incubate the cells at 37 °C for 2-5 min until the cells are detached and dispersed. NOTE: Tap the culture dish to detach the cells. View cells under the microscope to confirm complete detachment of cells from the surface of the dish. 

5. Add 5 ml complete media to the petri dish in order to neutralize trypsin. Pipette out the medium containing detached cells and collect it into a 15 ml centrifuge tube. 

6. Centrifuge the media containing endothelial cells at 220 x g for 5 min. 

7. Aspirate the supernatant and preserve the pellet containing cells. Suspend the pellet into 3-5 ml fresh rat brain endothelial cell growth medium by gently mixing it up and down with a pipette. Cells will be then be counted using automated cell counter or a hemocytometer. 

8. Transfer the cell suspension into fibronectin (50 μg/ml) precoated 8-well sterile chamber slide system, 0.7 sq. cm/well with a seeding density ranging between 10,000-15,000 cells per well. Grow the cells at 37 °C until confluence is achieved NOTE: For performing western blots or other experiments,cells can be grown in 10 cm cell culture dishes or special dishes as required by the experiment. 

2. Oxygen and Glucose Deprivation-Reoxygenation In Vitro Model 

1. Use the hypoxia cell culture system to study the effects of OGD-R on RBMECs (see Figure 1). Set up and calibrate the hypoxia cell culture system before beginning the experiment, according to the manufacturer’s instructions. 

2. Remove the confluent chamber slides (step 1.8) from the 37 °C incubator. Replace the complete medium in the chamber slide with deoxygenated, no glucose, Dulbecco's Modified Eagle's Medium (DMEM) and placed in hypoxia chamber with 95% nitrogen and 5% carbon dioxide for 2 h at 37 °C, to represent OGD condition. 

3. Move the cells back to the incubator with 95% oxygen, 5% carbon dioxide at 37 °C and provided with fresh rat brain endothelial cell complete medium and incubate for another 1 hr at 37 °C. 

NOTE: This step represents a reoxygenation situation.

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