Multi well glass bottom dish

In order to visualize acidic compartments, live cell imaging was performed for 661W cells stained with AO (R&D systems). 661W cells were plated in a multi-well glass bottom dish (Matsunami Glass Industry) at a density of 10,000 cells per well and incubated overnight at 37 °C. The medium was replaced with DMEM containing 1% FBS, and the cells were incubated with either 10 µg/mL MBE, 10 µM D3S5G or 10 µM D3G5G for 1 h at 37 °C, followed by exposure to blue light for 8 h. AO was added to a final concentration of 1 mg/mL, and the cells were incubated for 20 min at 37 °C. Finally, the medium was changed to prewarmed Live Cell Imaging Solution (LCIS, Thermo Fisher Scientific) containing 5.5 mM glucose and 1% FBS, and cells were subjected to live cell imaging using a 40× oil lens on an FV3000 microscope (Olympus Co.) equipped with a stage top incubator (Tokai Hit Co., Shizuoka, Japan). AO green and red fluorescence intensities were measured using ImageJ Ver. 1.53c (National Institutes of Health) for 24 cells each in four independent experiments to obtain the intensity ratio of green vs. red.

An open reading frame of Pep51 was amplified using gene-specific primers with extensions to vector ends (Fw; 5’-CCGGAATTCGCCATGTTGAGCATAAAATC-3’, and Rv: 5’- AAAGCGGCCGCTTAGCACAGTTTATTTG-3’, including stop codon), which contain an EcoRI site and NotI site, respectively. The amplified PCR product and the expression vector pcDNA4/V5 (Thermo Fisher Scientific, Waltham, MA, USA) were digested with EcoRI and NotI restriction enzymes, followed by ligation using Takara Ligation Mighty Mix (Takara, Shiga, Japan) according to the manufacturer’s instructions. The expression vector, empty pcDNA4 vector (0.1 μg), or transfection medium alone was transiently transfected into 5×10⁴ African green monkey kidney fibroblast cells (COS-7 line) or genetically engineered human embryonic kidney cells (HEK293MSR line) in 9.5-mm wells of multi-well glass-bottom dish (Matsunami, Osaka, Japan) with Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. COS-7 cells and HEK293MSR cells were grown under 5% CO₂ at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, and DMEM supplemented with 10% heat-inactivated FBS and 1% non-essential amino acids, respectively. Transfection efficiency (approximately 60-70%) was evaluated by an immunocytochemistry as described below.

The monocytic cell line THP-1 was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37 °C in 95% air and 5% CO₂. The cells were incubated in a multi-well glass-bottom dish (Matsunami Glass, Osaka, Japan) at a concentration of 1 × 10⁵ cells/100 μL in medium supplemented with 200 nM phorbol 12-myristate 13-acetate (Cayman Chemical, Ann Arbor, MI, USA) to induce differentiation into macrophage-like cells. Following 48-h incubation, the cells were washed with serum-free RPMI 1640 and cultured a further 12 h. Thereafter, cells were exposed to supernatant from human neutrophils or 100–300 mU/mL hNE (Innovative Research, Novi, MI, USA) in the presence or absence of SSH (100 µg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 6 h under serum-free conditions.