Alpha minimal essential medium

Neutralization titers to SARS-CoV-2 were determined as previously described (15 (link), 29 (link)). Briefly, 100 plaque-forming units of SARS-CoV-2 (2019-nCoV/USA_WA1/2020), Alpha, Beta, Gamma, Delta, and Omicron (BA.1, BA.2, BA.2.12.1, BA.4, and BA.5) was used on Vero-TMPRSS2 cells. The GISAID (global initiative on sharing all influenza data) IDs of Omicron live viral isolates used in neutralization assays are EPI_ISL_7171744 (BA.1), EPI_ISL_8818457 (BA.2), EPI_ISL_11685455 (BA.2.12.1), EPI_ISL_12416220 (BA.4), and EPI_ISL_13512579 (BA.5). Purified mAb was serially diluted threefold in duplicate starting at 10 μg/ml concentration in a 96-well round-bottom plate and incubated for 1 hour at 37°C. This antibody-virus mixture was transferred into the wells of a 96-well plate that had been seeded with Vero-TMPRSS2 cells the previous day at a concentration of 2.5 × 10⁴ cells per well. After 1 hour, the antibody-virus inoculum was removed, and 0.85% methylcellulose in 2% fetal bovine serum (FBS) containing Dulbecco’s minimal essential medium was overlaid onto the cell monolayer. Cells were incubated at 37°C for 16 to 40 hours. Cells were washed three times with 1× PBS (Corning cellgro) and fixed with 125 μl of 2% paraformaldehyde in PBS (Electron Microscopy Sciences) for 30 min. Following fixation, plates were washed twice with PBS, and 100 μl of permeabilization buffer was added to the fixed cells for 20 min. Cells were incubated with an anti–SARS-CoV spike primary antibody directly conjugated with Alexa Fluor 647 (CR3022-AF647) for up to 4 hours at room temperature.
Plates were then washed twice with 1× PBS and imaged on an ELISPOT reader (CTL Analyzer). Foci were counted using Viridot (counted first under the “green light” setting followed by background subtraction under the “red light” setting). IC₅₀ titers were calculated by nonlinear regression analysis using the 4PL (four parameter logic) sigmoidal dose curve equation on Prism 9 (GraphPad Software). Neutralization titers were calculated as 100% × [1 − (average foci in duplicate wells incubated with the specimen) ÷ (average number of foci in the duplicate wells incubated at the highest dilution of the respective specimen)].

The mouse endothelial cell line bEnd.3 was obtained from ATCC, Catalog number CRL-2299. MLO-Y4 was a gift from Dr Lynda F Bonewald. bEnd.3 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco Cat. 10099141) and 1% penicillin/streptomycin (P/S). MLO-Y4 cells were maintained in alpha–minimal essential medium (alpha­MEM) containing 10% FBS and 1% P/S. For 2D coculture, bEnd.3 cells were seeded on coverslips in 24-well plates. After 24 hours, MLO-Y4 cells were seeded in the wells that cultured bEnd.3 cells. The cells were maintained at 37 °C in a 5% CO2 humidified incubator.

Recombinant mouse proteins (Chi3l1, rIL13Rα2, RANKL, and M-CSF) were provided by R&D systems (USA). The Gibco BRL (USA) provided minimal essential medium alpha (α-MEM), fetal bovine serum (FBS), 0.25% EDTA-trypsin, and penicillin-streptomycin. RiboFECTTM transfection reagent, Chi3l1-siRNA, and IL13Rα2-siRNA came from RiboBio company (China). Primary antibodies targeting p-Akt (Ser473), Akt, p-ERK, ERK, p-JNK, JNK, p-P38, P38, p-P65, P65, and GAPDH were obtained from Cell Signaling Technology (CST, USA). The Abcam (USA) provided the primary antibody recognizing Chi3l1, which was used for immunofluorescence (IF) and western blot (WB). The primary antibody that recognized Chi3l1 used for co-immunoprecipitation (IP) and IL13Rα2 for IF and WB came from R&D systems (USA). Santa Cruz (USA) offered the primary antibody that recognized IL13Rα2 for IP.