Figure 1. Function and Nuclear Translocation Illustration of the Nrf-2 Pathway Preliminary data showed that SUL could ameliorate hyperoxia-compromised phagocytic function. This experiment sought to explore the mechanism behind this phenomenon. We hypothesized that the activation of Nrf-2 would increase the antioxidant potential of macrophages exposed to hyperoxia by induction of antioxidant protein, HO-1, leading to a reduction in iROS and the subsequent prevention of HMGB1 release. Significant advances in this field will have the potential to provide clinical and therapeutic approaches for patients suffering from hyperoxia and VAP.
MATERIALS AND METHODS
Cell Line Procedure Murine macrophage RAW 264.7 cells were cultured in Dulbecco’s …show more content…
After allowing the cells to grow for 24 h, they were washed once with PBS and treated with hyperoxia (95% O2). OptiMEM media was used to treat the cells. The supernatant, consisting of the media that the cells were grown in, was collected and placed into labeled 15 mL falcon tubes. Next, the tubes were centrifuged for 5 minutes in 4°C at 1000 rpm. 1800 µL of the supernatant was collected from falcon tubes and transferred to labeled centricons. Centricons were centrifuged for 40 minutes in 4°C at 4200 rpm. Centricons were removed and the liquid in the top cone was collected and transferred to labeled 500 mL Eppendorf tubes. Lastly, the samples were stored in -80°C until further …show more content…
Adherent cells were scraped and centrifuged at 500 x g for a 5 minutes. Next, cells were washed by suspending the pellet in PBS. 1-10 x 106 cells were transferred into a micro centrifuge tube and were centrifuged at 500 x g for approximately 2 minutes. Using a pipette, the supernatant was disposed, allowing the cell pellet to become dry. Cold CER I was applied to the cell pellet. For the cytoplasmic and nuclear protein extraction portion of the procedure, the first step consisted of suspending the cell pellet by vortexing it at the highest setting. The tube was incubated for approximately 10 minutes and following incubation, cold CER II was applied to the tube. Then, the tube was vortexed for 5 seconds and left on ice for 1 minute. The tube was vortexed once more for 5 seconds and was centrifuged at the highest speed for 5 minutes in a micro centrifuge. The supernatant was taken from the tube and placed in a new tube. The pellet fraction, containing the nuclei, was suspended in cold NER. The tube was vortexed for 15 seconds and was later placed on ice and vortexed for 15 seconds every 10 minutes for a total of 40 minutes. The tube was then centrifuged at maximum speed for 10 minutes in a micro centrifuge. The supernatant was placed into a clean tube on ice. The sample was stored at -80°C until further
MATERIALS AND METHODS
Cell Line Procedure Murine macrophage RAW 264.7 cells were cultured in Dulbecco’s …show more content…
After allowing the cells to grow for 24 h, they were washed once with PBS and treated with hyperoxia (95% O2). OptiMEM media was used to treat the cells. The supernatant, consisting of the media that the cells were grown in, was collected and placed into labeled 15 mL falcon tubes. Next, the tubes were centrifuged for 5 minutes in 4°C at 1000 rpm. 1800 µL of the supernatant was collected from falcon tubes and transferred to labeled centricons. Centricons were centrifuged for 40 minutes in 4°C at 4200 rpm. Centricons were removed and the liquid in the top cone was collected and transferred to labeled 500 mL Eppendorf tubes. Lastly, the samples were stored in -80°C until further …show more content…
Adherent cells were scraped and centrifuged at 500 x g for a 5 minutes. Next, cells were washed by suspending the pellet in PBS. 1-10 x 106 cells were transferred into a micro centrifuge tube and were centrifuged at 500 x g for approximately 2 minutes. Using a pipette, the supernatant was disposed, allowing the cell pellet to become dry. Cold CER I was applied to the cell pellet. For the cytoplasmic and nuclear protein extraction portion of the procedure, the first step consisted of suspending the cell pellet by vortexing it at the highest setting. The tube was incubated for approximately 10 minutes and following incubation, cold CER II was applied to the tube. Then, the tube was vortexed for 5 seconds and left on ice for 1 minute. The tube was vortexed once more for 5 seconds and was centrifuged at the highest speed for 5 minutes in a micro centrifuge. The supernatant was taken from the tube and placed in a new tube. The pellet fraction, containing the nuclei, was suspended in cold NER. The tube was vortexed for 15 seconds and was later placed on ice and vortexed for 15 seconds every 10 minutes for a total of 40 minutes. The tube was then centrifuged at maximum speed for 10 minutes in a micro centrifuge. The supernatant was placed into a clean tube on ice. The sample was stored at -80°C until further