D in a chemical hood for 3 hours. Three wash steps were

D in a chemical hood for 3 hours. Three wash steps were then performed. For each wash step, the microparticle solution was centrifuged at 4 , 4 krpm, for 5 minutes, and then the supernatant was removed. Subsequently, 40 mL of refrigerated water was added, the microparticle pellet was resuspended and the washing steps were repeated. After the last centrifugation step, 5 mL of water was added. Samples were snap frozen in liquid nitrogen and immediately placed in a lyophilizer. Following lyophilization, all microparticles were stored at -20 . For release and in vivo studies, an appropriate amount of microparticles were weighed out and suspended in an appropriate amount of PBS to reach the desired concentration. SEM imaging of microparticles and ImageJ quantification Lyophilized particles were placed on carbon tape (Electron Microscopy Sciences, Hatfield, PA) placed on aluminum mounts. Samples were sputtered with gold-palladium, and SEM imaging was performed with a LEO/Zeiss FESEM at the JHU School of Medicine MicFac. Microparticle loading and release profiles Microparticles were prepared as described with 10 or 100 of the peptide labeled with FITC. Loading efficiency was quantified by dissolving the microparticles in DMSO and adding to PBS. The solution was centrifuged to separate out the PLGA precipitate and the supernatant was collected for fluorescence measurement. For release studies, microparticles were diluted in PBS at 40 mg/mL in a 1.5 mL tube and incubated at 37 with light shaking. At the specified time points, samples were vortexed, spun down, supernatant was collected, and new PBS added to the microparticle pellet.Aliskiren hemifumarate DMSO was added to the supernatant so that the final solution for fluorescence measurements was constant 5 v/v DMSO/PBS.Astegolimab Fluorescence measurements were obtained using a BioTek Synergy 2 plate reader with an excitation filter of 485 +/- 20 nm and an emission filter of 528 +/- 20 nm.PMID:23659187 Peptide concentration was obtained by comparison to a standard curve for 6001-FITC in 5 v/v DMSO/PBS. In vitro assays for determination of peptide effects Human retinal endothelial cells (HRECs) (all cells used were P8-P12) were tested in three separate assays. SP6001’s effect on HREC apoptosis was tested by the caspase-glo 3/7 assay purchased from Promega (Madison, WI). Cells were plated at 5,000 cells/well in opaque 96well plates to minimize well-to-well cross-talk. After 24 h, complete endothelial cell media was replaced with serum free media. Next, media with 30/10 ng/mL (bFGF/VEGF) was added with or without peptide at 10 . After 48 h, caspase-glo luminescent reagent was added at 100 /well, and luminescence measured with a Victor V plate reader (Perkin Elmer). The experiment was repeated twice.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptBiomaterials. Author manuscript; available in PMC 2014 October 01.Shmueli et al.PageWe used the ACEA cell migration assay to assess SP6001 effect on cell adhesion, SP6001 was added to complete endothelial cell medium at 12.5 , and cells allowed to adhere in special E-plate (Roche, IN), suitable for cell culture with sensing electrodes. Impedance, correlated to cell adhesion, was measured using a RT-CIM system (ACEA Biosciences, Inc., San Diego, CA). HRECs were trypsinized and plated at 25,000 cells/well. Cells settled for 30 minutes before being loaded into the ACEA machine. Values are scaled to percent increase above the negative control (complete endothelial cell me.