Hacat cells

The cytotoxic potential of acneic and non acneic strains of C. acnes grown in BHI, RCM, or SLM was determined by the measurement of lactate dehydrogenase (LDH) release by HaCaT keratinocytes due to cytoplasmic membrane destabilization. HaCaT cells (CLS, Eppelheim, Germany) were cultured at 37°C in 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium (DMEM, Lonza) containing 25 mM glucose and supplemented with 10% inactivated fetal bovine serum, 2 mM L‐glutamine (Lonza), and antibiotics (penicillin 100 IU/mL and streptomycin 100 μg/mL). Cells were used between passages 41 and 65. They were seeded in 24 well plates at a final density of 5 × 10⁵ cells per well, and grown for 48 hr before use. A minimum of 8 hr before interaction with bacteria, cells were starved of antibiotics and fresh serum‐free medium was added. Bacteria were harvested (7,000×g; 10 min), washed in PS and HaCaT cells were infected at a bacterium‐to‐cell ratio of 50:1. The amount of LDH released by HaCaT cells was determined after 18 hr of incubation using the Cytotox 96 enzymatic assay (Promega, France) (Picot et al., 2003). Control studies performed using bacteria alone showed that none of the strains used in this study produced metabolites interfering with the assays.

Gel strength 90–110 gelatin A (GA, average 20–25 kDa) and trifluoroacetic acid (TFA) were purchased from Sigma–Aldrich (St Louis, MO, USA). Sodium alginate (SA), acetic acid, and acetonitrile (ACN) were purchased from Dae Jung (Gyeonggi-do, South Korea). Epidermal growth factor (EGF) was provided by Daewoong Pharmaceutical Co., Ltd. Dulbecco’s phosphate buffered saline (DPBS) was purchased from Welgene (Gyeongsangbuk-do, South Korea), Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Hyclone (Logan, UT, USA), 0.25% Trypsin-EDTA and fetal bovine serum (FBS) were purchased from Gibco (Paisley, UK).
HaCaT cells were purchased from Lonza (Walkersville, MD, USA). The cell counting kit-8 (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Human EGF antibody as primary antibody (R&D systems, Minneapolis, MN, USA), rabbit anti-mouse horseradish peroxidase (HRP) as secondary antibody (Abcam, Cambridge, UK) were purchased.