Figure 13. HIF-1a level in ischemic EC. (A) HIF-1a production was measured in EC treated for different time under ischemia. Cells were grown on collagen coated cover glasses and placed on the top of the ischemic vascular bed of chick embryo. RNA was isolated from the ischemia treated EC using RNA extraction kit. Loading of total RNA for cDNA conversion was normalized by running a gel for total RNA and calculating the band intensities for each set. After amplification, the band intensities from the representative images were calculated and plotted as a graph. Ischemia induced HIF-1a expression in EC in a time dependent manner. **P,0.001 significantly different than 0 min. (n = 5) (B) RL was administered to evaluate its effect on ischemia induced HIF- 1a expression by EC. EC grown on collagen coated cover glasses were placed on ischemic chick embryo vascular bed followed by RNA extraction from the EC using RNA extraction kit. Loading of total RNA for cDNA conversion was normalized by running a gel for total RNA and calculating the band intensities for each set. RL attenuated ischemia induced HIF-1a expression by EC. **P,0.001 versus ischemic. (n = 5). doi:10.1371/journal.pone.0010524.g013 EAhy926 cells and H9C2 cells were grown in collagen coated cover glasses for overnight at 37uC/5%CO2. Equal numbers of cells were seeded on cover slips according to the experimental need. Next, the cover glasses containing the cells were placed on top of the ischemic vessels thus allowing the cells to be in direct contact with the ischemic area. Measurement of free glutathione using CMFDA Measurement of GSH was performed using the CMFDA fluorescence probe as mentioned else where [54]. Ischemia was created in the blood vessels by following the protocol as mentioned earlier followed by incubating the cells on the top of the ischemic vessels. For RL based recovery experiment, vessels were treated with RL during the period of ischemia while cells were pretreated with RL for 15 min followed by incubation on RL treated ischemic vessels. Both tissues and cells were washed with PBS and incubated with CMFDA for 30 min. Next, the unbound CMFDA was washed of by washing with PBS for 2 times. Fluorescence Chick Embryo Ischemia Model images of the CMFDA bound tissues and cells were taken using Olympus IX71 microscope adapted with a DP71 camera. Next, fluorescence intensities of the images were calculated using Adobe Photoshop version 7.0. Measurement of superoxide using NBT Superoxide in ischemic vessels and cells were measured using NBT. Briefly, partial ischemia was created in the vessels by blocking the blood flow while the ischemic vascular bed was used to treat the cells under ischemia. Next, vessels and cells were washed with PBS and incubated with NBT for 2 h. For imaging, the vessels were washed with PBS and the images of the NBT stained vessels were taken with Nikon Cool Pix camera adapted to a stereo microscope. For colorimetric analysis of the sample, tissues and cells were homogenized and centrifuged at 6500 g to remove the debris. Absorption of the supernatant was taken in BioRad Elisa reader at 560 nm. Measurement of peroxynitrite using dihydrorhodamine DHR123 was used to quantify the level of peroxynitrite in ischemic vessels and cells. After ligation of the vessels, the chick embryos were kept for 2 h and the vascular bed was incised out using a sterile scissor. Tissues were washed with PBS and incubated with the DHR123 probe for 1 h. After 1 h incubation, the tissues and cells were washed with PBS and homogenized with a glass homogenizer. Homogenized samples were then centrifuged at 6500 g for 5 min and the supernatant was read at excitation/ emission of 500/536 nm. Hydrogen peroxide measurement using amplex red H2O2 level in ischemic vessels of chick embryo was measured using H2O2 specific fluorescence probe, amplex red. Ischemic vessels were incised out and washed with PBS for 3–4 times. Next, the tissues were incubated with amplex red for 30 min. For imaging, tissues were washed with PBS for 2–3 times and fixed on glass cover slips and the images were taken under 4X objective of Olympus IX71 microscope. For fluorimetric analysis, tissues were homogenized and centrifuged. The reading of the supernatant was taken at excitation/emission of 570/585 nm. Nitric oxide measurement using DAF-2DA fluorescence probe NO levels in the ischemic chick embryo vessels were measured using the DAF-2DA fluorescence probe. Ischemic vessels were excised out and washed with PBS. Next, the vessels were incubated with DAF-2DA and 1 mM L-arginine mixture for 20 min. After washing, the DAF-2DA probed vessels were fixed on the glass cover slide and imaged under 4X objective of Olympus IX71 microscope. The fluorescence intensity from the vessels was measured using the Adobe Photoshop version 7.0. Lipid peroxidation using TBA protocol We evaluated lipid peroxides by measuring TBARS using a technique modified from Bromont et al., 1989 [18]. Ischemic vascular bed and cells were homogenized in 5vol ice-cold 0.05 M phosphate buffer (pH 7). 1 ml buffer solution and 1.5 ml trichloro- acetic (TCA)-thiobarbituric acid (TBA)-HCl reagent prepared in 0.85N HCL with 13.5% wt/vol TCA and 0.33% wt/vol TBA and deoxygenated by bubbling with nitrogen were added to 0.2 ml homogenate. Samples were then placed in sealed tubes containing nitrogen and were heated for 15 minutes in a boiling water bath. After cooling, 1 ml TCA 70% wt/vol was added, and the precipitate was removed by centrifugation at 2,500 g for 10 PLoS ONE | www.plosone.org 12 May 2010 | Volume 5 | Issue 5 | e10524