Trypsin neutralizing solution

The differentiated AEC-ALI cells grown in 24-well plates were pre-treated, or not, for 16 h with PI (4 mg/mL) and infected apically with RV-A16 at three various doses (1, 2.5, 5 (×10⁵ pfu/well)) at 4 °C (to enable virus binding to the ICAM1 receptor but to block virus entry to cells) or 35 °C (to enable virus binding and entry) for 2 h with gentle swirling. The cells were washed with DPBS 3 times before lysis with RLT buffer (Qiagen, Germantown, MD, USA) for RNA extraction or treated with 300 µL of 0.25% Trypsin/EDTA (Gibco, Waltham, MA, USA) for 5 min at 35 °C to dissociate the cells and cleave cell surface proteins, including ICAM-1. After adding 600 µL of trypsin-neutralizing solution (Lonza, Walkersville, MD, USA), the cells were collected by pipetting into Eppendorf tubes and spun down at 200× g for 5 min following washing with 1 mL of DPBS twice. The cell pellets were lysed by adding RLT buffer for RNA extraction. RNA samples were used to determine RV-A16 viral RNA copy numbers using qRT-PCR.

To measure the binding of rhLK8 to the GRP78 protein on the surface of HUVECs, rhLK8 was labeled with FITC using a FITC-labeling kit (Sigma) according to the manufacturer’s instructions. HUVECs that had been transduced with scrambled siRNA or GRP78-specific siRNA were trypsinized and neutralized by trypsin neutralizing solution (Lonza). 5×10⁵ HUVECs were stained using FITC-labeled rhLK8 or GRP78 antibodies for 1.5 h at 4°C, washed with PBS 3 times, and analyzed by flow cytometry with a FACS Caliber (BD Biosciences).

To investigate the effect of blockage of E-cadherin or the responsiveness to cytokines of NP epithelial cells, detached epithelial cells from a normal ethmoid and NP were harvested and cultured. Epithelial cells were resuspended in bronchial epithelial cell growth medium (BEGM, containing bovine pituitary extract, insulin, HC, GA-1000, retinoic acid, transferrin, triiodothyronine, epinephrine and hEGF, Lonza, USA) and cultured at 37°C in a 5% CO₂ atmosphere. Upon reaching 80% confluence, cells were trypsinized, neutralized by trypsin neutralizing solution (Lonza, USA), and subcultured in BEGM. Upon reaching confluency, the subcultured epithelial cells from normal ethmoid were incubated with 1 or 5 μg/ml of anti-E-cadherin neutralizing antibody (DECMA, Santa Cruz Biotechnology, USA) and the subcultured epithelial cells were treated with either 100ng/ml of Th1 cytokines, TNF-α and IFN-γ, or of Th2 cytokines, IL-4 and IL-5 (PeproTech, USA). After overnight incubation, protein or RNA was extracted from the control and treated cells as detailed above.

Cell viability was measured by trypan blue exclusion assay. Before the assay, cells were rinsed with warmed phosphate buffer solution pH 7.4 (PBS, Thermo Fisher Scientific) and then trypsinized with 0.5% polyvinylpyrrolidone (Sigma-Aldrich) in trypsin/EDTA 0.025% solution (Lonza) for 6 min at 37 °C. Then, trypsin-neutralizing solution (Lonza) was added following by centrifugation at 130 rpm for 5 min. The cell pellet was resuspended in 5 ml of fresh cell media. Cell viability was estimated using 0.6 ml of cell suspension in a Vi-Cell XR viability analyzer (Beckman Coulter).
Cellular membrane damage was measured by detection of lactase dehydrogenase (LDH) in the cellular medium using a colorimetric assay (LDH Cytotoxicity Detection Kit, Takara Bio). Absorbance of the assay was measured at 491 nm for 30 min at 25 °C using a Cytation 3 plate reader with constant shacking (Biotek).

Trypsin with EDTA, trypsin neutralizing solution, endothelial cell growth medium-2 (EGM-2) with hydrocortisone, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, GA-1000, heparin, and fetal bovine serum (FBS), were purchased in Lonza (Basel, Switzerland). Empagliflozin (11575), dapagliflozin (11575), and canagliflozin (11575) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA) and 25-OHC was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used for immunofluorescence: VE-Cadherin (D87F2) XP^® Rabbit mAb #2500 were purchased from Cell Signaling (Warszawa, Poland), and anti-Rabbit (H+L) Alexa Fluor Plus 488 was purchased from Invitrogen (Waltham, MA, USA).