Dmem cell culture media

The ADAM10 and ADAM17 moderately-specific FRET enzyme substrate PEPDAB005 (green-color fluorescence with optimal excitation and emission wavelengths of 485 nm and 530 nm, respectively) 21 (link), 22 (link) was obtained from BioZyme Inc (Apex, NC). Cell-membrane selective lipophilic fluorophore DiD (near-infrared fluorescence with optimal excitation and emission of 644 nm and 665 nm, respectively), was purchased from ThermoFisher Scientific (Pittsburgh, PA) as a Vybrant cell-labeling solution. Cell-nuclear DNA specific Hoechst 33342 fluorescent dye (blue fluorescence with optimal excitation and emission of 350 nm and 461 nm, respectively) was purchased from ThermoFisher Scientific. Phycoerythrin (PE)-conjugated mouse monoclonal antibodies (mAb) to human ADAM10 and ADAM17 and corresponding isotype control mAb (red-color fluorescence with excitation and emission wavelengths of 564 nm and 573 nm, respectively) were obtained from R&D Systems (Minneapolis, MN). Paraformaldehyde (PFA) (10% aqueous solution) and Glutaraldehyde (GAL) (2.5% solution in 0.1 M Millonig's Sodium Phosphate Buffer, pH 7.2) were purchased from Electron Microscopy Sciences (Hatfield, PA). RPMI-1640 and DMEM cell-culture media, fetal-calf serum (FCS), Trypsin and enzyme-free cell-dissociation solution were obtained from GIBCO-Life Technologies (Grand Island, NY).

B16 and 293 T cells were cultured in DMEM cell culture media (Gibco, Carlsbad, CA), supplemented with 1% Penicillin–Streptomycin (Gibco, Carlsbad, CA) and 10% fetal bovine serum (Life Technologies, Burlington, ON). Cell lines were routinely checked for Mycoplasma contamination using a PCR kit (Abm, Cat#: G238). PCSK9, D374Y, and Q152H cDNA plasmid were produced by Dr. Seidah and subcloned into pLNCX-geneticin retroviral plasmid and subsequent transfection was performed following our published conditions [49 (link)].

HepG2 cells were obtained from the National Centre for Cell Science, Pune, India (local repository for the American Type Cell Culture (ATCC)), proliferated within two passage numbers, and stored in liquid nitrogen at passage number 17. Cells were cultured in high glucose (4.5 gm/L) DMEM cell culture media (Gibco, Evansville, IN, USA) supplemented with 10% FBS (HiMedia, Mumbai, India) and 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and maintained at 37 °C and 5% CO₂ [63 (link)]. Cells were passaged at 70% confluence and used for assays based on the number of cells required in each experiment. All experiments were conducted within 5 passages.

Both A549 lung cancer and normal HFF cells were seeded (5 × 10³ cells/cm²) and cultured for 24 h in a complete cell culture medium containing (DMEM cell culture media (Gibco), 10% FBS, streptomycin (100 mg/mL) and penicillin (100 U/mL at standard conditions (37 °C, 95%humidity, 5%CO₂ ventilation). The cultured cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and cultured for 24 h at standard conditions. The cultured cells were exposed to a range of CHG-NPs doses (31.2, 62.5, 125, 250, and 500 μg/mL). Following 48-h exposure, the old media was refreshed with fresh media containing MTT (0.5 mg/mL). After a further 3-h incubation at 37 °C, DMSO was added to the wells to dissolve the produced formazan. The concentration of produced formazan was estimated by recording the sample's absorbance at 570 nm as the cells' survival index (Stat fax 2100 plate reader). The cells' viability was measured as the following equation:

RAW 264.7 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Pentane, ipopolysaccharide, and λ-carrageenan were purchased from Sigma-Aldrich (St Louis, MO, USA), and DMEM cell culture media, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco BRL (Invitrogen Co., Carlsbad, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). The NO detection kit and PRO-PREP™ were purchased from iNtRON (Seongnam, Republic of Korea). Absorbance was measured using a device purchased from Molecular Devices (Sunnyvale, CA, USA). The RNA isolation kit was purchased from Qiagen (Santa Clarita, CA, USA). The reverse transcription master mix was purchased from Elpis Biotech (Daejeon, Republic of Korea). All primary antibodies, iNOS, COX2, TNF-α, IL-6, IL-1β, Erk1/2, p-Erk1/2, JNK, P-JNK, P38, p-P38, NFκB, P-NFκB, and β-actin, were purchased from Cell Signaling (Denver, MA, USA). iNOS and COX-2 enzyme-linked immunosorbent assay (ELISA) kits were purchased from MyBioSource (San Diego, CA, USA), and the IL-6 and IL-1β ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

In this experimental study, for virus packaging,
HEK293T cells were grown in DMEM cell culture
media (Gibco, USA) supplemented with 10% fetal bovine
serum (FBS), 100 units/ml penicillin (Pen), 100 mg/ml
streptomycin (Strep, all from Gibco, USA) and incubated
in 37°C with 5% CO₂. To passage, HEK293T cells were
separated from flask by Trypsin-EDTA (Gibco, USA)
and after two passages, HEK293T cells with confluency
of about 70-80% were used for virus packaging. PsPAX2
plasmid comprising of the gag/pol packaging genes and
pMD2.G plasmid composed of VSV-G were co-transfected
with pLenti-III-miR-GFP-has-miR-124 (also pLenti-IIIGFP-
mir-control vector) by calcium phosphate transfection
method, as previously described (Fig .1A, B) (17 (link)). Viral
supernatant was collected every 12 hours post-transfection
until 72 hours, and it also was centrifuged (3000×g for 10
minutes at 4°C) to remove cell debris. Finally, viruses were
concentrated using ultracentrifugation at 21000 rpm at 4°C
for 3 hours. Viral titration was performed on HEK293T
cells with a serial dilution of the viral stock. Virus stock was
aliquoted and it was frozen at -70°C for further use.