Fluorostar optima plate reader

Cell viability was measured using the Cell Counting Kit-8 (CCK8; Dojindo Molecular Technologies, Gaithersburg, Maryland, USA). Briefly, wild-type TPC-1 cells, and TPC cells stably expressing either non-targeting shRNA or LGR5 shRNA were plated and cell growth measured over 48 hours. Absorbance measurements were made by the FluoroStar Optima plate reader (BMG Labtech) at excitation filter 465 nm. Measurements were performed in triplicate.

Enrichment-ELISA is a method to identify enrichment of target binding phages during phage display selection. All steps were carried out at RT. A total of 20 pmol/well of the biotinylated SARS-CoV-2 RBD target protein was immobilized on a streptavidin- functionalized polystyrene 96-well plate (High capacity, Thermo Fisher, USA). Both the target-immobilized and target-free surfaces were quenched with biotin as described previously. The surfaces were blocked with 1% (w/v) BSA in selection buffer for 1 h and washed with washing buffer (0.2% (w/v) BSA, 0.05% Tween-20 in selection buffer). A total of 2.5 × 10¹¹ phages from the TS input samples or library were diluted in 100 µL washing buffer and incubated on target and control surfaces for 1 h. After washing with 200 µL washing buffer, the anti-M13 gpVIII monoclonal HRP-antibody (Sino Biologicals, Beijing, CHN) was diluted to a final concentration of 0.4 mg mL⁻¹ in washing buffer and 100 µL per well was incubated for 1 h. After six-fold washing with washing buffer, 100 µL of 3,3′,5,5′-3,3,5,5-Tetramethylbenzidin (Sigma-Aldrich, St. Louis, MO, USA) substrate solution was incubated for 20 min and stopped by addition of an equal volume of 2 M H₂SO₄. The signal intensity was quantified by absorption measurement in a Fluorostar optima platereader at 450 nm (BMG Labtech, Ortenberg, GE; n = 3).

Neutrophil granulocytes were isolated from rats treated with PS, LO or LO+300 mg/kg SZR-72 24 h prior to AP induction using Ficoll-Hypaque density gradient centrifugation. After sacrifice, blood was collected in EDTA coated tubes from each animal. Blood was gently mixed with equal volume of 3% Dextran solution and left to sediment for 40 min. In a conical tube, the leukocyte-rich plasma was carefully added on top of Ficoll-Hypaque, forming two phases. After centrifugation (250 RCF, 40 min) polymorphonuclear and red blood cell pellet was obtained. Erythrocytes were lysed with 0.2 % NaCl solution for no more than 30 sec. Immediately thereafter, lysis was stopped with ice-cold 3% NaCl solution. If red color was still visible after centrifugation, the process was repeated. Granulocytes were resuspended in phosphate buffered saline (PBS) containing 10 mM glucose, then cells were counted in a Bürker chamber. Cell number was adjusted to 1.5x10⁴/100µl. H₂O₂ production was measured with Fluorostar Optima plate reader (BMG Labtech, Ortenberg, Germany) using Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit described by the manufacturer.