Enzyme linked immunosorbent assay elisa kit

The Broncho-Alveolar Lavage fluid (BALF) supernatant from mice was collected following centrifugation (4 min at 7000g) and stored at -80°C. Inflammatory cytokine levels (IL1-β, IL-6 and TNF-α) in the BALF were determined as per manufacturer instructions using Enzyme Linked Immunosorbent Assay (ELISA) kits obtained from Biolegend, Inc, (San Diego, CA).

HaCaT and NCTC 2544 cells were seeded at the concentration of 5 × 10³ cells/mL in a 96-well multiwell culture plate. When cells reached the 80% confluence, culture medium was removed and replaced with fresh culture medium containing or not SDS (50 µg/mL), TNF-α (50 ng/mL), pure DHA (10 and 30 µM), or DHA carried by SLNs at the same concentrations of pure DHA, alone or in combination. At the indicated time points (48 and 72 h), cell supernatants were collected, centrifuged to remove cell debris, and stored at −80 °C until analysis. The production of cytokines was performed by using commercially available Enzyme-linked immunosorbent assay (ELISA) kits (Biolegend, San Diego, CA, USA) following the manufacturer’s instructions. The minimum detectable amounts of IL-1β, IL-6, and MCP-1 were 0.5, 4, and 3.9 pg/mL, respectively.

For DC maturation experiments, the cytokines IL-10, TNF-α, IFN-γ, and IL-12p70 were assayed in culture supernatants with Biolegend enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. For lymphocyte proliferation assays, the cytokines IFN-γ, IL-4, IL-17A, IL-10, IL-12p70, and TNF-α were quantified in supernatants of PBMC cultures using Pro Human Cytokine Group 1 6-Plex 1 × 96 kit (Bio-Rad) on a Bio Plex^® 200 (Bio-Rad).

Crude E. cava flake (CEF) was obtained from MilaeML (St. Ogeum, Seoul, Korea). RAW264.7 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin were purchased from Gibco (Grand Island, NY, USA). LPS (Escherichia coli 0111:B4) was purchased from Sigma (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from RMS Bio-solution (Seoul, Korea). Thiazolyl Blue tetrazolium bromide (MTT) was purchased from Alfa Aesar (Ward Hill, MA, USA). Griess reagent was purchased from Sigma (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioLegend (San Diego, CA, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). PD098059 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Primers were purchased from Bioneer (Daejeon, Korea). Antibodies were purchased from Cell Signaling Technology (Boston, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The possessed human skin samples were transferred to new 12-well plates (one piece/well; 250 µL) containing growth media with lipopolysaccharide (LPS, 5 μg/mL) in the absence or presence of topically jojoba preparations applied, as is, or integrated with the emulsifier. Dexamethasone (10 µM), supplemented in the media, was used as a positive control for a reduction of inflammation. After 48 h, the viability of the epidermal layer, separated by heat (56°C; 1 min), was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as previously reported (Wineman et al., 2015 (link)). The spent media from all test groups were collected, centrifuged, aliquoted and stored at −80°C until use. In addition, a similar set of treated skin pieces was fixed by formaldehyde (4%) for 24 h at 2–8 °C. Then, the tissues were washed twice with PBS and transferred to 70% ethanol until used. Following dehydration, 10 µm paraffin sections were prepared, and slides were stained with hematoxylin-eosin (H&E) solution, as previously described (Ogen-Shtern et al., 2020 (link)).
Measurements of specific markers of inflammation, interleukin (IL)-6, IL-8 and TNFα, in the collected spent media were quantified by commercial Enzyme-Linked Immunosorbent Assay (ELISA) kits (Biolegend, San Diego, CA).

All
of the chemicals were obtained from Sigma-Aldrich
unless otherwise specified. The solvents for chemical reactions were
distilled before use. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(poly(ethylene glycol))₋₂₀₀₀] (DSPE-PEG-MAL) and sulfhydrylated lactoferrin (Lf-SH) were purchased
from Laysan Bio, Inc. (Arab, AL). Milli-Q water was supplied by a
Milli-Q Plus System (Millipore Corporation, Bedford). RPMI 1640 culture
medium, penicillin–streptomycin, and fetal bovine serum (FBS)
were purchased from Gibco-BRL (Grand Island, NY). Primary antibodies
and fluorescent secondary antibodies for CLSM imaging were provided
by Abcam and Invitrogen, respectively. Enzyme-linked immunosorbent
assay (ELISA) kits were provided by Biolegend.