3550 uv microplate reader

Cell proliferation was evaluated in 96‐well plates using the tetrazolium compound MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl) 2‐(4‐sulphophenyl)‐2H–tetrazolium, inner salt] from Promega (Madison, WI, USA). Briefly, melanoma cells (800‐1000/well) were dispensed into flat‐bottom 96‐well plates and grown at 37°C in a 5% CO₂ humidified atmosphere. For chemosensitivity assay, cells were exposed to graded concentrations of vemurafenib (PLX4032; Hoffmann‐La Roche Ltd, Basel, Switzerland). Vemurafenib was dissolved in DMSO and, just before use, diluted to the appropriate concentrations in complete medium with final DMSO concentration never exceeding 0.05% (v/v). Six replica wells were used for each condition in a total volume of 100 μL. After 5 days, 20 μL of MTS solution was added to each well and cells were incubated at 37°C for 1‐3 hours. Absorbance was read at 490 nm (reference wavelength 655 nm) using a 3550‐UV Microplate reader (Bio‐Rad, Hercules, CA, USA). Chemosensitivity was measured in terms of IC₅₀, that is the concentration of the drug capable of inhibiting cell growth by 50%.
To evaluate cell doubling time, MTS was added to cells at different time‐points (0, 24, 48 and 72 h) after seeding (800‐1000 cells/well).

Conditioned media from melanoma cell lines were obtained by semi‐confluent cell cultures after incubation for 24 hours in 0.1% BSA/RPMI‐1640 medium without FBS. These conditions did not significantly affect cell viability. Culture supernatants were collected and concentrated at least 10‐fold in Centriplus concentrators (Amicon). Cells were detached from the flasks with a PBS/EDTA solution and the total cell number/culture was recorded to normalize cytokine secretion. The amounts of VEGF‐A and PlGF present in the culture medium were determined using commercial ELISA kits (R&D Systems), following the manufacturer's instructions. Optical density at 405 nm was measured in a 3550‐UV Microplate reader (Bio‐Rad).

Colons were flushed with cold PBS, opened along longitudinal axis, cut into 0.5 cm pieces and incubated for 24 h in RPMI 1640 supplemented with 10% FCS and antibiotics (n = 3–5 mice per group). Colon supernatants were collected and stored at −20°C. TNF-α serum levels were detected using a mouse TNF-α ELISA kit (Biolegend) according to the manufacturer's protocol. Mouse cytokines and chemokines were analyzed using Bio-plex Pro Mouse Cytokines 23-plex Assay (Bio-Rad) according to the manufacturer's protocol. IFN-γ levels in sera or colon supernatant were measured using an ELISA. Rat anti-mouse IFN-γ (Molecular Immunology, HZI) in coating buffer were incubated in 96 well plates (MaxiSorb TM Immunoplates, Nunc) overnight at 4°C. The 96 well plates were then blocked for 1 h, with 3% BSA in 0.05% Tween 20. Diluted sera or colon supernatant were distributed to the wells and incubated for 2 h at room temperature. IFN-γ was detected with biotinylated anti-mouse IFN-γ antibodies (Molecular Immunology, HZI). Biotinylated antibodies were bound with horseradish peroxidase (HRP) conjugated streptavidin (BD Pharmingen). Bound HRP was determined using o-Phenylendiamin (OPD) as substrate and the results were read using an ELISA-reader (BioRad 3550-UV microplate reader) at a wavelength of 490 nm.

Cell proliferation was evaluated by Cell Counting Kit-8(CCK-8; Dojindo, Kumamoto, Japan). Briefly, 5 × 10³ cells of LL2 cells were plated in 96 well plates and were incubated with either 0.1% (v/v) DMSO (control) or various concentrations of SB225002 (S7651, Selleck, a potent, and selective CXCR2 antagonist with > 150-fold selectivity over CXCR1) for 24 h, 48 h and 72 h at 37 °C. After treatment, cells were incubated in 10% CCK-8 reagent for another 2 h. The OD value was measured at 450 nm with a microplate reader from Bio-Rad (Microplate reader 3550-UV). For apoptosis assay, 5 × 10⁵ cells were plated in 6-well plates and incubated with culture medium in the presence of DMSO or various concentrations of SB225002. After 24 h, cells were harvested and washed twice with cold PBS. Those cells were stained with Annexin V/propidium iodide (PI) (BD Biosciences) and examined by NovoCyte Flow Cytometer (ACEA Biosciences) and data was analyzed by NovoExpress® software (1.3.0, ACEA Biosciences). According to TdT-mediated dUTP Nick-End Labeling (TUNEL) assay kit (Promega), treated cells were plated in 24-well plates on coverslips and stained with TUNEL reagent and DAPI. The coverslips were then moved on slides after washed with PBS. TUNEL staining was analyzed with a fluorescence microscopy (Eclipse 80i; Nikon Co., Tokyo, Japan).

Semi-confluent tumor cell cultures were incubated in 0.1% BSA/RPMI-1640 medium without FBS for 24 h. Culture supernatants were collected, centrifuged at 600× g for 10 min to remove cells in suspension, concentrated at least ten-fold in Centriplus concentrators (Amicon, Beverly, MA, USA) and frozen at −20 °C till use. Cells were detached from the flasks with a solution of 1 mM EDTA in PBS and the total cell number/culture was recorded. Quantification of the amount of VEGF-A in the concentrated supernatants was performed using Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark) coated with goat anti-VEGF-A IgGs, as previously described [31 (link)]. Briefly, detection of the cytokines was performed with biotinylated goat anti-VEGF (R & D Systems, Abingdon, UK) and streptavidin-alkaline phosphatase conjugate (1:10,000) (Roche). The reaction was stopped and optical density at 405 nm was measured in a Microplate reader 3550-UV (Bio-Rad, Hercules, CA, USA).
Modulation of VEGFR-2 phosphorylation in response to VEGF-A in untreated cells or cells exposed to EA was analyzed using the PathScan^® Phospho-VEGFR-2 (Tyr1175) Sandwich Elisa Kit (Cell Signaling Technology, Danvers, MA, USA).