4.3. RAW 264.7 Cells

The murine macrophage cell line, RAW 264.7, was obtained from American Type Culture Collection (ATCC TIB-71, Manassas, VA, USA). The RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Cape Town, South Africa) supplemented with 10% heat inactivated fetal bovine serum (FBS, Hyclone, Little Chalfont, UK), glutamax (Sigma-aldrich, St. Louis, MO, USA), antibiotic/antimycotic (Sigma-aldrich) and gentamicin (Sigma-aldrich). The cells were incubated in a humidified atmosphere of 5% CO₂ at 37 °C and the cells were sub-cultured every 2–3 days.

The RAW 264.7 cells (1 × 10⁵ cells/mL) were cultured in cell culture treated 48 well plates and incubated in a humidified atmosphere of 5% CO₂ at 37 °C for approximately 48 h until the cells reached 80–90% confluence. After the incubation period, media was removed and replace with media containing 2.5% FBS. The subsequent procedures occurred in serum free media. The cells were pre-exposed for 2 h to various concentrations of GONPs. Thereafter the cells were left unstimulated and a positive control was also present. The positive control were cells only stimulated by lipopolysaccharide (LPS) (1 µg/mL) without the presence of nanoparticles and this would mimic an inflammatory immune response. The final concentration of FBS/well was 0.5%. Cultures were incubated overnight (~18 h) under standard tissue culture conditions. Culture supernatants were collected for nitric oxide (NO), interleukin 6 (IL-6), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2 and proteome profiling analysis.

After the removal of the supernatants, cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS) (Lonza), supplemented with glutamax, antibiotic/antimycotic solution. Cytotoxicity was measured by adding 150 µL of a 1/10 dilution of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1, Roche, Basel, Switzerland) reagent in serum free medium to each well. Metabolically active cells convert WST-1 reagent to a formazan that can be measured spectrophotometrically. Formazan formation was determined by reading the plate at 450 nm (Multiskan Ex, Thermo Electron Corporation, Waltham, MA, USA) immediately after WST-1 addition and again after an incubation period of 1 h at 37 °C. The increase in absorbance at 450 nm is proportional to formazan formation. The level of fomazan formed is directly proportional to cell viability.

After the overnight incubation of the RAW 264.7 cells, the amount of nitrite that was produced by the cells was measured in the culture supernatant as an indication of NO production. The NO assay is based on the Griess reaction [31]. The amount of NO production was measured against a doubling dilution range of a 100 μM nitrite standard (Sigma-aldrich). Nitrite standards or culture supernatant collected (100 µL) were mixed with 100 µL of Griess reagent (1:1 of 1% sulfanilamide and 0.1% naphtylethlemidimine-dihydrochloride in 2.5% phosphoric acid) (all reagents obtained from Sigma-aldrich). Thereafter, the plate was incubated at room temperature for 15 min. The absorbance was read at 540 nm by a microplate reader (Multiskan Ex, Thermo Electron Corporation) and the amount of NO produced by the RAW cells quantified.

The mouse IL-6 ELISA (e-Bioscience, Ready-Set-Go, Waltham, MA, USA) kits were used to measure IL-6 cytokine levels in the cell culture supernatants. The LPS stimulated control was assayed at (1/40) while the negative control (not treated with LPS) was assayed at (1/5) in assay diluent. Assays were performed in 96 well Nunc maxisorb plates. The kit contained all the reagents for the assay and was performed as per the manufacturer’s instructions (see addendum).

Mouse MIP-1α, MIP-1β and MIP-2 ELISAs (R & D Systems, Minneapolis, MN, USA) were performed on the samples and the LPS stimulated culture supernatants. The kits contained all the reagents required for the experiment and experiments were performed as per the manufacturer’s instructions. The samples were all diluted in reagent diluent, 1% human serum albumin (HSA) (w/v). The MIP-1α samples were assayed at 1/270 and the LPS stimulated supernatant at 1/10,000. For the MIP-1β ELISA, the unstimulated culture supernatants were assayed at 1/20 while the LPS stimulated supernatants were assayed at 1/5000. The MIP-2 ELISA unstimulated supernatants were assayed at 1/100 and the mitogen stimulated supernatant at 1/1000.

A commercially available antibody array kit (Proteome Profiler, Mouse cytokine Array Panel A, R & D Systems) which was coated with 40 capturing antibodies in duplicate on a nitrocellulose membrane (dot blot) was used. The kit contained all the reagents for the assay and was performed as per the manufacturer’s instructions. This cytokine and chemokine antibody array was used to determine the effects of GONPs exposure on cytokine and chemokine by RAW 264.7 macrophage cells.The assay required 500 μL of cell culture supernatants (unstimulated 0 μg/mL GONPs, LPS stimulated 0 μg/mL GONPs and unstimulated 15.6 μg/mL GONPs). Membranes were subjected to an ultra-sensitive chromogenic 3,3′,5,5′-Tetramethylbenzidine (TMB) membrane substrate (Thermo Scientific, Waltham, MA, USA) to reveal sample-antibody complexes labeled with streptavidin-HRP. Photographs were taken of the blots after the exposure to the substrate.

Membrane images were quantified using image processing and analysis Java software(version 1.6.0_24, Oracle Corporation, Redwood city, CA, USA), ImageJ (version 1.4.3.67, National Institutes of Health, Bethesda, MD, USA). Levels of cytokines and chemokines were expressed as a percentage of the reference spot. Microsoft Excel was used to calculate the percentages which is expressed as mean ± standard deviation (SD).