Biotin conjugated rabbit anti fd bacteriophage

Supernatants from 96-well bacterial culture plates (see above) were used for initial HCA screening. DU145 cells were seeded in 96-well plates (BD Biosciences) overnight, and incubated with phage-containing supernatants and 50 μg/ml TexasRed-conjugated 70-kDa neutral dextran (ND70-TR, Invitrogen) in DMEM/10% FBS at 37 °C with 5% CO₂ overnight. Cells were washed 3× with PBS, fixed with 4% paraformaldehyde (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS for 10 min, washed 3x in PBS, and then permeabilized in PBS containing 1% fraction V bovine serum albumin (Fisher Scientific) and 0.1% TritonX-100 (Sigma). Phage were detected with 3.5 μg/ml biotin-conjugated, rabbit anti-fd bacteriophage (Sigma-Aldrich) for 1h at RT followed by 1 μg/ml Alexa Fluor® 488-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) for 15 min at RT. Hoechst 33342 (Thermo Scientific) at 1 μg/ml for 30 min at RT was used to detect nuclei. The 96-well plates were imaged on a CellInsight™ NXT HCS platform (Thermo Scientific) with a semi-aprochromat 20× LUCPLFLN objective (Olympus) utilizing >6 fields per well with a minimum of 300 cells per well. Pearson's correlation coefficient analysis between ND70-TR and phage particles were conducted using Thermo Scientific HCS Studio software suite on all imaged fields and averaged per well.