After the isolation (2.5.4.3), primary keratinocytes were seeded on a plastic or glass surface, coated with collagen I and fibronectin (Col/FN) or a LN5-rich matrix (LN), and cultured in a special medium at 34°C in 5% CO2 air with 95% humidity. The growth medium was changed
one day after seeding and every 2-3 days afterwards. Keratinocytes were grown without splitting until control cells became fully confluent (7-10 days).
Keratinocyte Medium:
MEM ... 500 ml [Sigma #M8167] Chelated FCS... 40 ml (8%)
Insulin (5 mg/ml in 4 mM HCl)...500 µl (5 μg/ml) [Sigma #I5500]
EGF (200 μg/ml)* ...5 µl (10 ng/ml) [Sigma #E9644] Transferrin (5 mg/ml)* ... 1000 µl (10 μg/ml) [Sigma #T8158]
10 mM Phosphoethanolamine*...500 µl (10 μM) [Sigma #P0503]
10 mM Ethanolamine*...500 µl (10 μM) [Sigma #E0135]
Hydrocortisone (5 mg/ml in EtOH) ..36 µl (0.36 μg/ml) [Calbiochem #386698]
200 mM L-Glutamine (100x)... 5 ml [Gibco # 25030-024]
Penicillin/streptomycin (100x)... 5 ml [PAA Laboratories #P11-010]
→ medium was sterile filtered (0.22 µm) before adding antibiotics
→ marked compounds (*) where diluted in PBS
Before adding to the medium, FCS (Gibco #10270-106) was depleted of Ca2+ ions, which induce differentiation of keratinocytes. Chelex resin (Biorad #142-2832) was suspended in 5 l ddH2O (40 g/l) and adjusted to pH 7.4 with HCl. Afterwards the suspension was filtered through
a paper filter (Schleicher&Schuell #314856) and the buffered resin was gently stirred with 500 ml of a heat inactivated FCS, O/N at 4°C. Chelex beads were removed by filtering and subsequent centrifugation (5 min, 8000 rpm). FCS was sterilized with a vacuum filter system (Corning #431097), aliquoted (40 ml) and stored at -20°C.
2.5.4.2 Surface coating
The Col/FN-coated surface was prepared by incubating dishes/plates/chamber slides with the coating medium for 2-4 h at 37°C. Afterwards, containers were directly used or sealed with Parafilm (ANC Co) and stored at 4°C. Before seeding cells, coating medium was removed and the dishes were washed with PBS.
Coating medium
MEM ...(21.5 ml) 25 ml [Sigma #M8167]
BSA, pH 7.0 (1 mg/ml)* ... 2.5 ml (0.1 mg/ml) [PAA Laboratories #K41-001-100] 1 M Hepes, pH 7.3 ... 500 μl (20 mM) [Sigma #H0887]
Vitrogen-collagen I (3 mg/ml) ...250 µl (30 µg/ml) [Cohesion #FXP-019/Nutacon #5409] Fibronectin (1 mg/ml)... 250 μl (10 µg/ml) [Invitrogen #33016-015]
100 mM CaCl2* ... 290 μl (1 mM)
→ fibronectin was diluted in sterile ddH2O
→ marked compounds (*) where diluted in PBS and sterilized by filtration (0.2 µl)
The LN matrix was deposited by RAC-11P/SD squamous cell carcinoma cells (Sonnenberg
et al., 1993) and prepared according to a previously published protocol (Delwel et al., 1993), with modifications. In short, cells were seeded in a dish/plate/chamber slide and grown until confluency in D-MEM containing 10% FCS. Afterwards, cells were washed three times with PBS and incubated O/N at 4°C with sterile 20 mM EDTA/PBS solution supplemented with protease inhibitor cocktail tablets (cOmplete, Mini, EDTA-free tablets; Roche #11836170001). The following day, cells were removed as a single sheet by short pipetting (along the walls of the dish). The matrix was washed once and stored in PBS at 4°C until use.
2.5.4.3 Isolation of primary keratinocytes from adult mice
Isolation of epidermis keratinocytes from older mice was carried out according to a modified protocol of Romero and colleagues (Romero et al., 1999). Sacrificed mice were shaved and sequentially washed in an iodine solution, distilled water and 70% EtOH (~60 s in each). After limbs were cut off, the skin was peeled off from the trunk and placed in a sterile Petri dish (10 cm Ø). The subcutaneous layer was removed very thoroughly with a scalpel (scraped off), as any remaining fat would drastically reduce the activity of trypsin. Skin was cut into fragments (~3-5 cm2) and placed (epidermis down) in a dish filled with 25 ml of antibiotics-containing PBS
for 5-10 min. Afterwards, skin fragments were transferred (epidermis up) to a new dish filled with 25 ml of 0.8% trypsin/PBS (Trypsin 1:250; Invitrogen #27250-018) and incubated for 50 min at 37°C. Epidermis was separated with forceps, transferred to the DNase medium (MEM supplemented with 0.25 mg/ml DNase I [deoxyribonuclease; Sigma #DN25] and 8% chelated FCS; 2.5.4.1; sterile filtered – 0.2 µm) and incubated with shaking for 30 min at 37°C in a water bath. Afterwards, the suspension was filtered through a 70 µm cell strainer (BD Biosciences). Collected cells were spun down (5 min, 500g) and washed once with the DNase medium. Subsequently cells were resuspended in the growth medium (2.5.4.1), counted and seeded on a coated surface (2.5.4.2) at a density of ~6×106 cells per dish (10 cm Ø), 1-1.5×106 cells per well (6 well plate) and 2-4×105 cells per chamber slide well.
Antibiotics-PBS
PBS ... 500 ml
Penicillin/streptomycin (100x)... 10 ml [PAA Laboratories #P11-010] Nystatin (10000 U/ml)... 10 ml [Sigma #N1638]
Fungizone (250 µg /ml)... 10 ml [Gibco # 15290-026]
2.5.4.4 Immunofluorescence staining of cells
Cells intended for IF analysis were grown on glass chamber slides (Nunc) coated with a suitable substrate. At chosen time points they were briefly rinsed in PBS and fixed for 10 min in freshly prepared cold 3.7% PFA/PBS. A permeabilization with 0.1% Triton X-100/PBS was performed for 15 min before 60 min blocking with 2% BSA/PBS. Alternatively Triton X-100 was added to the blocking solution. After rinsing with PBS, primary antibodies were applied and incubated for 60 min at RT. The rest of staining procedure was performed as described for skin IF (2.7.2.1). Slides were dried O/N at RT and stored at 4°C. Staining results were analysed using a fluorescence microscope (DM RA2; Leica) and archived with a digital camera (OrcaER; Hamamatsu).
2.5.4.5 Adhesion assay
Wells for the adhesion assay (96-well plate) were coated with substrates used for standard keratinocyte culture. The Col/FN coating was obtained by incubating a surface with PBS containing 30 µg/ml collagen I and 10 µg/ml fibronectin O/N at 4°C. The LN matrix was prepared as described in chapter 2.5.4.2. Before seeding cells, plates were washed with PBS, blocked with 1% BSA/PBS (PAA Laboratories) for 90 min at 37°C and washed again.
After isolation from adult mice, keratinocytes were resuspended in a standard growth medium (2.5.4.1) without FCS and seeded in 100 µl at a density of 105 cells/well. Three maximal value samples were transferred to a 1.5 ml tube (3×105 cells in 300 µl). Cells seeded on the plate
were incubated for 1-2 h at 37°C. Afterwards non-attached cells were removed by washing 3 times with PBS and 50 µl of substrate buffer were added to each well. Keratinocytes in the tube were spun down, resuspended in 150 µl of the substrate buffer and transferred to 3 empty wells (50 µl/well). The plate was incubated O/N at 37°C. The following day, 75 µl of the stop buffer were added to each well and the absorbance was measured at 405 nm with a plate reader (Dynatech MR5000). Substrate buffer A: NPAG (Sigma #N9376)... 25.7 mg 0.1 M Na-citrate, pH 5.0... 10 ml B: Triton X-100...0.5 %
→ both buffers prepared in ddH2O
→ A + B mixed [1:1] and stored at -20°C
Stop buffer
Glycine, pH 10.4... 50 mM EDTA... 5 mM
→ prepared in ddH2O
2.5.4.6 Time-lapse video microscopy and cell spreading analysis
Live cell recordings of freshly isolated keratinocytes (from 8-months-old mice) were performed using a Zeiss Axiovert 200 M microscope with a motor-controlled CCD camera (OrcaER, Hamamatsu). Images of cells were captured every 20 min for 42 h (at 37°C, 5% CO2).
The recording was started immediately after keratinocytes were seeded on Col/FN coated plastic (6-well plates). The cell size for spreading analysis was assessed by measuring the pixel area of 30-40 randomly picked cells in pictures taken at indicated time points. Evaluation and processing of images were performed with the Metamorph software (Universal Imaging Corporation).
2.5.4.7 Isolation of primary keratinocytes from 3d old mice
Epidermis of 3-day-old mice was isolated according to a modified protocol previously described by Caldelari and co-authors (Caldelari et al., 2000). In short, mice were sacrificed using ether (10-15 min) and washed in an iodine solution and 70% EtOH (60 s in each). After limbs and the tail were cut off, the skin was peeled off and placed, dermis up, in a Petri dish (6 cm Ø) filled with 4-5 ml of 10 µg/ml gentamicin/PBS solution (stock: 5 mg/ml; Sigma #G1397). The washing step was performed for 5-10 min, twice. Subsequently, the skin was transferred, epidermis up, to a dish containing 4-5 ml of dispase-II solution and incubated O/N at 4°C.
The following day epidermis was separated from dermis and torn into pieces using forceps. Fragments were placed in 15 ml tube with 10 ml of 5x trypsin/EDTA solution (Gibco #15400-054; 2.5.1.2) and incubated for 10 min at 37°C. During the trypsinization, the suspension
was mixed a few times to keep the tissue pieces afloat. Afterwards, part of the cell suspension (~5 ml) was transferred to a new tube, avoiding the epidermis pieces. K-SFM medium containing 10% FCS (5 ml) was added to the remaining part, mixed and transferred again. Cells were spun down for 5 min at 1000g, resuspended in the medium for counting and washed with PBS before the lysis.
Dispase II solution:
K-SFM (not supplemented) ... 45 ml [Gibco #10744-019]
Dispase II... 0.25 g (5 mg/ml) [Roche #165859]
Penicillin/streptomycin (100x)... 5 ml [PAA Laboratories #P11-010]
→ solution was sterile filtered (0.2 µm) before adding antibiotics;