Ufc901008

Adult porcine bladders were acquired (Tissue Source, LLC, Lafayette, IN) and urinary bladder extracellular matrix (ECM) was isolated as previously described [17 (link)]. ECM sheets were then lyophilized for 72 hours, comminuted through a 40-mesh sieve in a Thomas Scientific Wiley Mini Mill, and tumbled in a Fisher Scientific tube revolver in normal saline at 10 mg/mL for 24 hours at room temperature. ECM particles were pelleted via centrifugation at 100,000 rpm for 30 minutes and supernatant was passed through 100 um cell strainers and then 0.22-micron sterile filters. Resulting liquid was concentrated 200-fold by volume in 10kDa cutoff columns (MilliporeSigma UFC901008, Darmstadt, Germany) and protein concentration was approximated using a Nanodrop A280 measurement to verify cutoff column filter integrity. Samples were stored at −20°C until use.

Cells were obtained by peritoneal lavage of C57BL/6 mice. Briefly, mice were euthanized and injected i.p. with 5 ml ice-cold PBS using a 24-gauge needle, and the peritoneal lavage fluid was then captured using the same syringe. Cells were washed in complete medium and cultured in 12- or 6-well non-TC-treated cell culture plates (Corning, Corning, NY) for 12–48 hr. In some experiments, medium was supplemented with recombinant cytokines (Peprotech, Rocky Hill, NJ) and/or blocking antibodies as indicated. In other experiments, medium was supplemented with conditioned medium from tumor cell lines, which was filtered through a 0.22 uM filter to remove cellular debris. B16-conditioned medium was concentrated 20X using a 10 kDa centrifugal filter unit (cat #UFC901008, Millipore-Sigma). After culture, cells were washed to remove suspension cells, and macrophages were lifted by vigorous pipetting of ice-cold PBS. Macrophages were identified as live F4/80 + cells by flow cytometry.

The 2 ml aliquot was chromatographed on a peptide-affinity column (peptide corresponding to epitope 4 of CTR and conjugated (linker maleimidocaproyl-N-hydroxysuccinimide) to thiopropyl sepharose 6B ~4 ml bed volume, prepared by Mimotopes (Clayton, VIC, Australia). Once loaded, the pump was stopped for 5 min to allow binding and then the affinity column was washed with PBS. The breakthrough contained unbound conjugate in which the binding determinants were most likely compromised. The bulk of the conjugated material bound to the column was eluted with 100 mM glycine buffer (pH 2.2) and 1 ml fractions were collected into 100μl TRIS buffer (pH 9). Active fractions were concentrated with Amicon Ultra-15 centrifugal filter devices-10 K MWCO (15 ml: EMD-Millipore UFC901008), washed with PBS/1 mM EDTA (prefiltered 0.22 μm) and concentrated to ~1 ml. Using the Nanodrop1000, the λ1 (560 nm) and λ3 (280 nm) were recorded and used to calculate the amount of protein, the percentage of recovery and the degree of labelling (DOL) with AF568:NHS esters for the antibody conjugate. The final DOL was 4.4 and the yield 36%. The protein profile was checked on an 8% acrylamide gels stained with Coomassie (not shown). This overall protocol as outlined for the conjugation of mAb2C4 produced consistent labelling of active mAb2C4:AF568.