Fluorescence Localization of Tight Junction Proteins and Stress Fibers in Endothelial Cells

Protokół

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 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 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 cm²/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 (OGD-R) In Vitro Model

1. Use the hypoxia cell culture system to study the effects of OGD-R on RBMECs in (see Figure 1). Set up and calibrate the hypoxia cell culture system before beginning the experiment, according to 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%, N₂ and 5% CO₂ for 2 h at 37 °C, to represent OGD condition.
3. Move the cells back to the incubator with 95% O₂, 5% CO₂ 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.
4. Use the chamber slides for immunofluorescence localization and rhodamine phalloidin labeling (see section 3).

3. Immunofluorescence Localization of zonula occludens-1 (ZO-1) and filamentous actin (f-actin) Labeling Using Rhodamine Phalloidin

1. Expose the chamber slides containing RBMEC monolayers to 100 μl of opti-MEM/reduced serum media/well for 1 hr. Wash the chamber slides 3 times in 100 μl of phosphate-buffered saline (PBS, pH 7.0-7.2).
2. Fix the cells using 100 μl of 4% paraformaldehyde in PBS (pH 7.0-7.2) for 15 min and wash the chamber slides for 3 more times in PBS (pH 7.0-7.2).
3. Permeabilize the cells using 100 μl of 0.5% Triton X-100 in PBS, (pH 7.0-7.2) for another 15 min. Block with 100 μl of 2% bovine serum albumin (BSA) in PBS for an hour. After this step, stain/label cells either for ZO-1 or f-actin.
   1. For immunofluorescence staining incubate the cells with an anti-rabbit primary antibody against ZO-1 in 1:150, prepared in 2% BSA-PBS for overnight (O/N) at 4 °C. Wash the cells 3 times in PBS (pH 7.0-7.2). Incubate with 100 μl of Fluorescein isothiocyanate (FITC)-tagged anti-rabbit secondary antibody for 1 hr at room temperature (RT).
   2. For Rhodamine Phalloidin labeling, following blocking, expose the cells to 100 μl of rhodamine phalloidin in 1:50 dilution, prepared in 2% BSA-PBS, for 20 min.
   3. Wash the cells from immunofluorescence staining and rhodamine phalloidin labeling in PBS, (pH 7.0-7.2). Mount the chambers using mounting media containing anti-fade reagent with 4′,6-diamidino-2-phenylindole (DAPI).
4. Visualize the cells using a 60 X water immersion lens and cells are scanned in a single optical plane under a confocal microscope.

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