Materials and Methods

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Cell culture conditions and maintenance

To investigate the eicosanoid response of human cells, two immortalised cell lines were selected (HaCaT keratinocytes and 46BR.1N fibroblasts). Immortalised cell lines were used as they offer unlimited lifespan, reproducible results and unrestricted quantities, due to the fact they indefinitely proliferate (Segrelles et al., 2011). However, the modification to the genetic code of the cells can result in aberrant functions and behaviour (Lewis et al., 2006), therefore all results need to be confirmed with primary cells. Here, immortalised skin cell lines were treated with various inflammatory stimuli and used to validate the corresponding model predictions.

Cell lines

Human adult low-calcium high-temperature (HaCaT) keratinocytes were purchased from CLS Cell Lines Service GmbH. This is a non-tumorigenic, spontaneously transformed, immortal human epidermal keratinocyte cell line which displays no major functional defects (Boukamp et al., 1988). The recommended cell culture medium for HaCaT keratinocytes was DMEM containing 10% FBS.

The 46BR.1N human fibroblast cell line was purchased from The European Collection of Cell Culture (ECACC) (Salisbury, UK). This cell line represents immortalised dermal fibroblasts, showing normal cell morphology and function (Arlett et al., 1988). The recommended cell culture medium for 46BR.1N fibroblasts was MEME containing 15% FBS, L-glutamine (200nM), non-essential amino acids (200 nM) and sodium pyruvate (200 nM).

Cells were seeded into plastic dishes, typically 75 cm2 culture flasks or 100 mm Petri dishes for UV experiments, and were grown in the recommended cell culture medium at 37°C, 5% CO2 and 95% humidity. The media was changed every 2-3 days depending on the cell growth, and to subculture the cells, they were washed with PBS (without Ca2+/Mg2+) and detached using trypsin/EDTA. The cell suspension was neutralised using an equal volume of serum-containing medium. Cells were centrifuged at 200xg for 3 min, and the pellet was reconstituted in the appropriate culture media. For maintenance, both cell lines were normally passaged at a dilution ratio of 1:2 three times a week.

Cell counting

Cell counting was performed automatically by using a TC20 cell counter. The reconstituted cell pellet (10 µL) was mixed with 0.4% trypan blue solution (1:1 dilution). The automated counter reading was expressed as a number of cells/mL, and the total amount of cells were calculated using the following equation.

Total number of cells (cells⁄mL)=Number of cells × Original volume (mL)

Cell treatments

UVR

Both cell lines were irradiated using a Herbert Waldmann 236 B (UV6) UV lamp (Germany). The lamp was switched on for at least 10 min prior to the experiment to allow it to warm-up. Directly before any experiment, the UV irradiance (mW/cm2) was measured using Variocontrol UV meter (Waldmann, Germany). The time of exposure was re-adjusted to the required dose using the UV irradiance and was calculated using the following equation.

UV Dose (mJ⁄cm^2 )=UV irradiance (mW⁄cm^2) × Time (s)

All cell types (80% confluent, grown in 100 mm Petri dishes) were washed with PBS and then fresh PBS (5 mL) was added. The lid of the Petri dish was opened and cells were irradiated with 15 mJ/cm2 UV dose. The PBS was then removed and cell-appropriate serum-free medium (10 mL) was added. Non-irradiated cells were subjected to the same procedure, but the UV lamp was switched off. The cells were incubated for the required amount of time (0.5, 1, 3 and 6 h).

A23187

A23187 treatments were prepared by dissolving commercially available A23187 (5 mg) in DMSO (955 µL) to a final 10 mM stock solution. The stock solution was stored at -20°C in 100 uL aliquots. Each aliquot was defrosted once and then disposed of. To fresh cell-appropriate serum free media, calcium chloride (1.8 mM) was added. Treatment media was prepared by directly pipetting A23187 (10 mM) into the culture medium at no more than 0.1% v/v of DMSO. For example, the 5 μM dose was prepared by adding 9 μL of A23187 (10 mM) to 18 mL of cell-appropriate serum free media. The control treatment was made by dilution of 9 μL of DMSO in to 18 mL of cell-appropriate serum free media.

Prior to treatment, both cell types were washed with PBS (5 mL/dish), and replaced with the cell-appropriate treatment media (18 mL/dish) and incubated for the required amount of time (0.5, 1, 3 and 6 h).

ATP

ATP treatments were prepared by dissolving commercially available ATP (551.1 mg) in water (10 mL) to a final 100 mM stock solution. The stock solution was stored at -20°C in 500 uL aliquots. Each aliquot was defrosted once and then disposed of. Treatment media was prepared by directly pipetting the ATP stock solution (100 mM) into the culture medium. For example, the 2 mM dose was prepared by adding 360 μL of ATP (100 mM) to 18 mL of cell-appropriate serum free media. The control treatment was made by dilution of 360 μL of water in to 18 mL of cell-appropriate serum free media.

Prior to treatment, both cell types were washed with PBS (5 mL/dish), and replaced with the cell-appropriate treatment media (18 mL/dish) and incubated for the required amount of time (0.5, 1, 3 and 6 h).

Calcium Ionophore and COX inhibited

Indomethacin treatments were prepared by dissolving commercially available indomethacin (17.9 mg) in DMSO (5 mL) to a final 10 mM stock solution. The stock solution was stored at -20°C in 100 uL aliquots. Each aliquot was defrosted once and then disposed of. Treatment media was prepared by directly pipetting the indomethacin stock solution (10 mM) into the cell-appropriate serum free culture medium at no more than 0.1% v/v of DMSO. For example, the 10 μM dose of indomethacin was prepared by adding 18 μL of indomethacin (10 mM) to 18 mL of cell-appropriate serum free media. The control treatment was made by dilution of 18 μL of DMSO in to 18 mL of cell-appropriate serum free media.

Prior to treatment, both cell types were washed with PBS (5 mL/dish), and replaced with the cell-appropriate treatment media (18 mL/dish) and incubated for 1 hour. The consequential A23187 treatments were prepared by dissolving commercially available A23187 (5 mg) in DMSO (955 µL) to a final 10 mM stock solution. The stock solution was stored at -20°C in 100 uL aliquots. Each aliquot was defrosted once and then disposed of. To fresh cell-appropriate serum free media, calcium chloride (1.8 mM) was added. Treatment media was prepared by directly pipetting A23187 (10 mM) into the culture medium at no more than 0.1% v/v of DMSO. For example, the 5 μM dose was prepared by adding 9 μL of A23187 (10 mM) to 18 mL of cell-appropriate serum free media. The control treatment was made by dilution of 9 μL of DMSO in to 18 mL of cell-appropriate serum free media.

After incubation, both cell types were washed with PBS (5 mL/dish), and replaced with the cell-appropriate serum free treatment media (18 mL/dish) and incubated for the required amount of time (0.5, 1, 3 and 6 h).

MTT Assay Method

Seeding density optimisation

In order to identify the exponential growth phase of HaCaT keratinocytes and 46BR.1N fibroblasts, eight seeding densities (0 – 2 x106 cells/well) were seeded into 6 well-plates in complete media. The cells were then incubated for two days in order to reach 80% confluency.

Dose optimisation of UVR

In order to assess the toxicity of UVR, cell lines were seeded into 6 well-plates in complete media until fully 80% confluent. Monolayers were then irradiated with 0-100 mJ/cm2 (for more details see section *) and cultured for 6h in serum-free culture media.

Dose optimisation of A23187

In order to assess the toxicity of A23187, cell lines were seeded into 6 well-plates in complete media until fully 80% confluent. Monolayers were then treated with 0-10 µM of A23187 (for more details see section *) and cultured for 6h in serum-free culture media.

Dose optimisation of ATP

In order to assess the toxicity of ATP, cell lines were seeded into 6 well-plates in complete media until fully 80% confluent. Monolayers were then treated with 0-32 mM of ATP (for more details see section *) and cultured for 6h in serum-free culture media.

Cell viability assessment

After seeding, treatment or irradiation, cells were washed with PBS. The MTT reagent (0.5 mg/mL) was added to each well (2.5 mL/well in a 6 well plate, 200 µL/well in a 96 well plate). The plate was returned to the incubator for 4 hours at 37ºC, 5% CO2, 95% humidity. The reagent was then aspirated and DMSO (2.5 mL/well in a 6 well plate, 200 µL/well in a 96 well plate) was added to each well to release formazan crystals. The plate was gently agitated for 10 min and 200 μL of each well content was transferred to a 96-well plate in triplicate. The blank sample was represented by DMSO to subtract the background reading. Absorbance (A) was measured at 540 nm. The mean absorbance of three technical repeats (triplicate wells) was regarded as one independent experiment. The percent of viability was assessed using the formula:

Viability (%)= ("Mean A of the sample - Mean A of the blank" )/("Mean A of the control - Mean A of the blank" )×100%