Ph d 12

Two different phage libraries encoding 12 amino acid peptides (PhD-12, New England Biolabs) and 20 amino acid peptides (TriCo-20, Creative Biolabs) were used in these experiments. TriCo-20 has a sequence diversity of 1.1 × 10¹⁰ and consisted of 20-amino acid peptides linked to pIII of M13 bacteriophage via a serine linker. PhD-12 has a titer of 2 × 10¹³ and consisted of 12-amino acid peptides connected to pIII of M13 bacteriophage via a (glycine)₃-serin linker. Four days after ischemia–reperfusion injury surgery, each phage library was administered intravenously via tail vein into two anesthetized animals. Animals were perfused through the right ventricular with at least 10 ml HBSS under anesthesia 15 min after injection to minimize non-specific binding of phages. Thereafter, ischemic LV, remote heart (the rest of the heart after removing ischemic LV and without aorta), kidney and liver were dissected and collected in PBS. The infarcted area was identified by discoloration, excised and all tissues were homogenized with a QIAGEN TissueLyser II and incubated with dispase II in RPMI medium for 40 min at 37 °C. Addition of RPMI medium (#11875093, Gibco) containing 10% fetal bovine serum (Gibco) stopped digestion and cell suspension was then transferred through a 100 µm mesh, followed by 30 µm mesh to exclude cell clumps.

Selection of specific peptides exposed on the surface of bacteriophages from the phage display libraries PhD-12 and PhD-C7C (New England Biolabs, Ipswich, MA) was carried out as described previously [21 (link)]. Briefly, approximately 10¹¹ phage particles from each library were pre-incubated with a non-specific mock mouse antibody and added to the wells of 96-well microtiter plates coated with 200 ng chFVN145 in PBS, pH 7.4, and incubated for one hour at 37°C. Then, unbound phage particles were washed away with PBST, and bound phages were eluted with 100 μg/mL chFVN145. The eluted phages were used for the second round of biopanning, which was carried out using plates coated with 20 ng chFVN145. Phage particles that were eluted after the second round were used to transfect E. coli ER2738 cells. Phages with the selected exposed peptides were isolated from individual plaques and assayed for their binding to chFVN145 by ELISA. For indirect ELISA, the wells of 96-well microtiter plates were coated with 20 ng of chFVN145 or 3% bovine serum albumin in PBS, pH 7.4. After blocking, individual selected phages were diluted in PBST to yield ~10¹⁰ colony forming units (CFU) and added to the wells. Indirect ELISA was performed with anti-M13 polyclonal rabbit antibodies followed by alkaline phosphatase-conjugated anti-rabbit IgG (ICN) and stained with p-nitrophenyl phosphate.