Spectramax 340pc

Macrophages were cultured on adherent plates for Bio-Plex assay (5 × 10⁴ cells/96-well) and analyzed for cytokine secretion 2, 6, and 24 h after LPS (100 ng/mL), or LPS (100 ng/mL) and IL-10 (100 ng/mL) treatment. Supernatants were collected for analyses and stored at −20 °C until use. Supernatants were analyzed for the presence of 27 different proteins with the Bio-Plex Pro™ human Cytokine 27-plex Assay Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.
For TNF-α secretion analyses, macrophages (5 × 10⁴ cells/96-well) were stimulated with LPS, or LPS and IL-10 for 6 h. Supernatants of stimulated macrophages were analyzed for TNF-α proteins with an enzyme-linked immunosorbent assay (ELISA) Kit (Invitrogen). The analysis was performed according to the manufacturer’s protocol with SpectraMax 340PC (Molecular Devices, San José, CA, USA).

To measure cell growth, cell viability was determined using a colorimetric assay (Cell Counting Kit-8; Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's instructions. A microplate reader (SpectraMax 340PC; Molecular Devices, Sunnyvale, CA) was used to measure the absorbance at 450 nm, and data were analyzed using Softmax Pro software (Molecular Devices). The relative growth rate was normalized to the control group.

Cell culture media was collected and soluble factors expression was analysed 24 h after initiation of cell culture. The levels of IL6, IL8/CXCL8, CCL2, IL1B, IL2, IL4, IL12, TNFα and IFNγ secreted into the growth medium were measured using Human IL-6 DuoSet ELISA Development Kit (R&D System, Wiesbaden, Germany), Human IL-8 Standard ABTS ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human CCL2/MCP-1 DuoSet (R&D System), Human IL-1β/IL-1F2 DuoSet ELISA Development Kit (R&D System, Wiesbaden, Germany), Human IL-2 ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human IL-4 Standard ABTS (PeproTech, Rock Hill, NJ, USA), Human IL-12 ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human TNFα ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human IFN-γ ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), and Human Standard ABTS ELISA Development Kit (Peprotech), respectively. The ELISA analysis was performed using high binding ELISA plates (Greiner BioOne) at RT according to the manufacturer’s instructions. Optical density was measured using photospectrometer Spectramax 340 PC (Molecular Devices) at the wavelength 450 nm.
The represented data show values of two independent analyses normalized to the levels of cytokine expression for cells grown without GAIN scaffolds.

Breast cells were seeded in 6-well plates and incubated with different bLf concentrations (50, 125 and 175 μM) for 24 and 48 h. Afterwards, cells were fixed for 90 min at −20°C in ice-cold 1% acetic acid in methanol, and then incubated with 0.5% (w/v) SRB in 1% acetic acid for 90 min at 37°C. After washing with 1% acetic acid and drying, protein-bound sulforhodamine B (SRB) was dissolved in 10 mM Tris for 10 min at room temperature (RT). A sample from each condition was transferred to a 96-well plate and absorbance was read at 540 nm in a microplate reader (SpectraMax 340PC, Molecular Devices). Results were normalized to the untreated cells, which were considered to have 100% cell proliferation.

For estimation of the chlorophyll and carotenoid contents, plant leaves were incubated in 80% (v/v) acetone for 1 h in the dark. The plant material was spun down and absorbances at 470, 645, and 663 nm were measured in a microplate spectrophotometer (SpectraMax 340PC; Molecular Devices). Total chlorophyll was calculated as 20.2A₆₄₅+8.02A₆₆₃ and total carotenoids were calculated as [1000A₄₇₀−1.82(12.7A₆₆₃−2.69A₆₄₅)−85.02(22.90A₆₄₅−4.68A₆₆₃)]/198 (Arnon, 1949 (link); Lichtenthaler and Buschmann, 2001 ).
Anthocyanin extraction and quantification was adapted from Ticconi et al. (2001) (link). Plant leaves were weighed (fresh weight, FW) and incubated at 100 °C for 5 min in extraction buffer composed of 1-propanol (37%, v/v), HCl, and H₂O, in a 18:1:81 ratio. Samples were subsequently incubated overnight at room temperature in the dark. The plant material was spun down and absorbance of the supernatant was measured at 535 nm and 650 nm in a similar microplate spectrophotometer. Total anthocyanins were calculated as A₅₃₅−A₆₅₀ g⁻¹ FW.