614 JOURNAL OF COSMETIC SCIENCE
for 1 hour. After a washing step, peroxidase-conjugated antibody against IL-6, IL1-RA
or hyaluronic acid were added. Plates were incubated at room temperature for 1 hour.
Samples were then incubated for 15 minutes at room temperature with a substrate solution
(containing H
2 O
2 ).Reaction was stopped and absorbance at 450 nm was measured using
a microplate reader.
HUMAN SEBOCYTES
Human sebocytes obtained with the reprogramming of induced pluripotent stem cell
technology (iPS) were provided by Phenocell (Grasse, France). Cells were routinely cultured
with 5% CO
2 at 37°C and 90% humidity atmosphere in regular Phenocult-SEB medium
(Phenocell, Grasse, France). Media were renewed every 48–72 hours to allow cell growth.
For experiments, cells were seeded at 2 × 104 cells/cm² into Phenocult-SEB medium until
confluency was reached. For IL-6 evaluations, cells were treated with pKTSKS peptide
(1–6 ppm) or 0.1% DMSO for 48 hours and with living planktonic C. acnes cells (300
bacteria: 1 sebocyte) for the last 24 hours. Then, cell culture media were collected, and
IL-6 quantification was performed as described in the keratinocyte section. Cell viability
was evaluated using nucleus labeling with Hoechst 33258 method (Sigma Aldrich,
Germany).24
HUMAN FIBROBLASTS ASSAYS
Human fibroblasts (NHF) from human foreskin were provided by CellnTechTM
(Switzerland). For routine maintenance, cells were seeded in 175 cm2 flask (Falcon™, New
York, USA) in DMEM, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 1µg/
mL fungizone, 4 mM L-glutamine, and 1mm sodium pyruvate (all Gibco®, Thermo
Fisher Scientific™, Illkirch-Graffenstaden, France). Cultures were incubated at 37°C in a
humidified atmosphere containing 5% CO
2 .The medium was renewed every 48 hours to
allow cell growth. NHF were used between passage 5 and 11.
For experiments, NHF cultures were seeded at a density of 2 × 104 cells/cm² in 24 wells cell
culture plates (Falcon™, New York, USA). Then, they were treated with various dosages of
peptide or its solvent (DMSO, 0.1% v:v, Merck) for 3 days in fresh medium. Cell culture
media were collected for ELISA studies and cell number was estimated using a Hoescht
33258 staining allowing the quantity of dermal proteins to be weighted to the number of
viable cells.
Collagen-I production was quantified in the culture media by measuring the carboxy-
terminal pro-peptide of procollagen type I (PIP) using the Takara MK101 assay. Fibronectin
production was measured using Takara MK115 kit. Samples were incubated in ELISA
plates coated with anti-PIP or anti-fibronectin antibody for 1 hour at 37°C, followed by
washing and incubation with peroxidase-conjugated antibody targeting the protein of
interest. The substrate solution (containing H
2 O
2 )was then added, and after 15 minutes
at room temperature, the reaction was stopped, and absorbance was measured at 450 nm.
For collagen-IV, R&D Systems LXSAHM-01 assays kit was used. Samples or standards
were incubated with the magnetic beads coated with an anti-collagen IV antibody, for
2 hours at room temperature under agitation. After washing, the beads were incubated with
for 1 hour. After a washing step, peroxidase-conjugated antibody against IL-6, IL1-RA
or hyaluronic acid were added. Plates were incubated at room temperature for 1 hour.
Samples were then incubated for 15 minutes at room temperature with a substrate solution
(containing H
2 O
2 ).Reaction was stopped and absorbance at 450 nm was measured using
a microplate reader.
HUMAN SEBOCYTES
Human sebocytes obtained with the reprogramming of induced pluripotent stem cell
technology (iPS) were provided by Phenocell (Grasse, France). Cells were routinely cultured
with 5% CO
2 at 37°C and 90% humidity atmosphere in regular Phenocult-SEB medium
(Phenocell, Grasse, France). Media were renewed every 48–72 hours to allow cell growth.
For experiments, cells were seeded at 2 × 104 cells/cm² into Phenocult-SEB medium until
confluency was reached. For IL-6 evaluations, cells were treated with pKTSKS peptide
(1–6 ppm) or 0.1% DMSO for 48 hours and with living planktonic C. acnes cells (300
bacteria: 1 sebocyte) for the last 24 hours. Then, cell culture media were collected, and
IL-6 quantification was performed as described in the keratinocyte section. Cell viability
was evaluated using nucleus labeling with Hoechst 33258 method (Sigma Aldrich,
Germany).24
HUMAN FIBROBLASTS ASSAYS
Human fibroblasts (NHF) from human foreskin were provided by CellnTechTM
(Switzerland). For routine maintenance, cells were seeded in 175 cm2 flask (Falcon™, New
York, USA) in DMEM, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 1µg/
mL fungizone, 4 mM L-glutamine, and 1mm sodium pyruvate (all Gibco®, Thermo
Fisher Scientific™, Illkirch-Graffenstaden, France). Cultures were incubated at 37°C in a
humidified atmosphere containing 5% CO
2 .The medium was renewed every 48 hours to
allow cell growth. NHF were used between passage 5 and 11.
For experiments, NHF cultures were seeded at a density of 2 × 104 cells/cm² in 24 wells cell
culture plates (Falcon™, New York, USA). Then, they were treated with various dosages of
peptide or its solvent (DMSO, 0.1% v:v, Merck) for 3 days in fresh medium. Cell culture
media were collected for ELISA studies and cell number was estimated using a Hoescht
33258 staining allowing the quantity of dermal proteins to be weighted to the number of
viable cells.
Collagen-I production was quantified in the culture media by measuring the carboxy-
terminal pro-peptide of procollagen type I (PIP) using the Takara MK101 assay. Fibronectin
production was measured using Takara MK115 kit. Samples were incubated in ELISA
plates coated with anti-PIP or anti-fibronectin antibody for 1 hour at 37°C, followed by
washing and incubation with peroxidase-conjugated antibody targeting the protein of
interest. The substrate solution (containing H
2 O
2 )was then added, and after 15 minutes
at room temperature, the reaction was stopped, and absorbance was measured at 450 nm.
For collagen-IV, R&D Systems LXSAHM-01 assays kit was used. Samples or standards
were incubated with the magnetic beads coated with an anti-collagen IV antibody, for
2 hours at room temperature under agitation. After washing, the beads were incubated with
