After each peptide alteration, a series of protease mutation and optimization cycles were performed

ted, 250 l of cellular supernatant containing monoclonal antibodies, anti-lymphocyte function associated antigen 1 and anti- Intercellular adhesion molecule 1 , were used along with 100 l of blocking buffer for 30 min at 4C. AntiE-Selectin was used at 1:200 dilution in the blocking buffer for 30 min at 4C. After thorough washing of cells post treatment, specific secondary antibodies tagged with FITC or PE to Lfa1 and Icam1 wherever necessary were diluted 1:200 in the blocking buffer and added to cells for 30 min at 4C. The cells were washed thoroughly and stained for nucleus using bisBenzimide H 33342 at 500 nM dilution for 15 min at 4C and fixed using 4% PFA for 10 min at 37C for microscopy only. Images were acquired using Zeiss 510 Meta confocal microscope. Wherever necessary, the amount of total fluorescence was obtained using Infinite 200 pro fluorescence microplate reader and represented as fold change with respect to untreated C57BL/6 control APECs. For fluorescence measurement and flow cytometry, cells were finally fixed using 4% PFA for 10 min at 37C and washed well with PBS before acquiring the same using BD FACS Verse and analyzed using WinMdi 2.8 software. Live cell imaging and motility Imaging of APECs was performed at 40X using Leica DMI6000B live imaging microscope at 37C along with 5% CO2. Images were acquired every 5 min and made into a video using the LAS AF software. The videos were analyzed using the manual tracking method plugged into the Image J software. Briefly, the motility of a minimum of 25 APECs were tracked across 6 h, i.e. 1824 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698015 h post treatment unless otherwise mentioned and represented as a single video. The changes in the X and Y coordinates in each image of the video for each cell were used to calculate their independent velocities. The average velocities exhibited by 5 / 28 Ifn and Nos2 Regulate Functions of APECs untreated C57BL/6 APECs were considered as 100% and the other conditions were represented in comparison to the same as percentages. Intracellular infection of APECs with SalGFP For the in vitro infection assay, 0.2 x 106 APECs were plated per well in 96 well plates in RPMI with 10% FCS without antibiotics. APECs were infected with an overnight culture of Sal-GFP at a multiplicity of infection of 1:50 for 45 min, unless otherwise mentioned. Cells were washed with PBS post infection and 100 g/ml gentamycin was added into the medium for 45 min. Cells were washed again with PBS and cultured in RPMI with 10% FCS containing 25 g/ml gentamycin. At 2 h post-infection, the CFU burden was elucidated and interpreted as phagocytic uptake by APECs. APECs were treated with or without 25 U/ml of Ifn for 24 h as well as in the presence or absence of LNMA post infection. The number of Sal-GFP per cell was elucidated by staining cells for Lamp1 and bisBenzamide H 33342 and counting the total number of Sal-GFP in each field and divided by the total number of cells in the field manually across different Z positions using a 63X magnification using the ApoTome.2 fluorescence microscope. For finer details, images were acquired at 100X magnification using a Zeiss confocal microscope. ELISA The amounts of cytokines, Ifn, Il6, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698359 Tnf, in the sera were estimated using ELISA kits from eBioscience, USA. Protocols recommended by the manufacturers were followed to quantify the amounts of cytokines in each sample. TMB was used as the substrate for the AGI 5198 site development and optical density readings were obtained at 450 nm using VersaMa