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<p><span><strong>PRIMARY MOUSE KERATINOCYTE CULTURES</strong></span></p> <p>Isolation of epidermal keratinocytes from neonatal mice is based on the protocol of Dlugosz et al., <em>Methods Enzymol</em>. 254:3-20 (1995). The epidermis from a newborn mouse should yield approximately 5-10x10<sup>6</sup> cells, with a 30-40% plating efficiency. </p> <ol><li>Sacrifice newborn mice by CO<sub>2</sub> narcosis. <u>Note:</u> Newborn mice may require 10-15 min in the CO<sub>2</sub> chamber. </li> <li>Sterilizing washes are performed in batch in a large beaker on ice, so that carcasses are completely submerged: <ol><li>Wash in 10% iodine solution in PBS for 10 min; decant. </li> <li>Rinse with sterile PBS (+pen/strep), then 70% ethanol, then wash in 70% ethanol for 10 min; decant. </li> <li>Rinse with sterile PBS, then keep mice in PBS (+pen/strep) on ice. </li> </ol></li> <li>Removal of skin is performed in a sterile environment. <ol><li>Remove the tail and prepare it for genotyping by PCR (see separate protocol). </li> <li>Remove limbs. (Optional: limbs can be used to prepare frozen skin sections). </li> <li>To remove skin from the torso and head, place the carcass on a sterile tissue culture dish. Use a sterile scalpel to make a longitudinal incision from tail to snout, then peel the skin from the carcass using sterile, curved forceps. Avoid puncturing the gut to maintain sterility. Layer the skin onto the bottom of a clean dish, with the dermis facing down, and spread out all edges. Allow the skin to dry for a short time (this can be done on ice). </li> </ol></li> <li>Use curved forceps to carefully pick up and place each skin, dermis facing down, onto 5 ml of cold, sterile 0.25% trypsin solution (GibcoBRL) in a 60mm tissue culture dish. Use the tips of the forceps to tease out edges that fold under, so that the skin is floating flat with most of the epidermis above the surface of the trypsin solution. Incubate at 4°C for 15 to 24 hrs. </li> <li>Transfer each skin to a dry, sterile tissue culture plate and spread it out with the epidermis facing down. Pull the dermis from the epidermis using a sterile pasteur pipette and discarded it (the dermis is a jelly-like blob; the epidermis is a thin, tissue paper-like sheet). </li> <li>Mince the epidermis and suspend it in 6 ml of growth medium using a 10 ml pipette. Transfer the suspension to a sterile 10 ml Falcon tube, and pump up and down to release keratinocytes from the epidermis. Pass the suspension through a sterile, 70μm nylon filter (Becton Dickinson) into a fresh 50 ml Falcon tube to remove cornified sheets. Rinse the initial tube with 5 ml fresh growth medium, and rinse it through the same filter into the same 50 ml tube (cells are in a final volume of 10-11 ml). </li> <li>Plate the entire 10 ml of keratinocyte suspension from each skin onto a 10cm tissue culture dish coated with ~30μg/ml denatured rat tail collagen (Vitrogen; Collagen Corporation). <dl><dd><u>To prepare collagen-coated dishes: </u><br>Dilute Vitrogen stock (~3 mg/ml) 1:100 in sterile PBS, then completely cover the surface of a 10cm dish with 3-5 ml. Incubate at 33°C for several hrs or at 4°C overnight. Completely aspirate collagen from the dish before adding cells. <u>Note:</u> Do not allow Vitrogen stock to warm, or it will begin to gel. </dd></dl></li> <li>Culture mouse keratinocytes at 33-34°C, 8% CO<sub>2</sub> for five to seven days before use in experiments. If necessary for some experiments, the cultures can be passaged once or twice for expansion.</li> </ol><p><span><strong>Growth Medium for Primary Keratinocytes</strong></span></p> <p>EMEM, Ca<sup>++</sup>-free (BioWhittaker catalog #06-174G), supplemented with: </p> <ul><li>4% FBS, chelated </li> <li>0.05 mM CaCl<sub>2</sub></li> <li>0.4 μg/ml hydrocortisone </li> <li>5 μg/ml insulin </li> <li>10 ng/ml epidermal growth factor (EGF) </li> <li>10<sup>-10</sup> M cholera toxin </li> <li>2x10<sup>-9</sup> M T3 </li> <li>100 units/ml penicillin and 100μg/ml streptomycin </li> <li>2 mM L-glutamine </li> </ul><p><span><strong>STOCK PREPARATIONS:</strong></span></p> <p><span><span><strong><em>FBS, chelex-treated</em></strong></span></span></p> <p>FBS is chelex-treated in batch with Chelex-100 resin (BioRad, cat # 1422832) to remove free Ca<sup>++</sup>. Use 100 g chelex resin per 500 ml FBS. Swell 100 g chelex resin in 400-500 ml distilled water, then titrate to pH 7.4 with HCl while stirring (pH will take a while to stabilize during titration). Filter through Whatman #1 paper. Scrape resin slurry into 500 ml FBS and stir at room temp for 3 hr or at 4°C overnight. Filter the chelated FBS through Whatman #1 paper and discard the resin slurry. Filter the chelated FBS through a 0.2μm bottle filter to sterilize it. Aliquot sterile, chelated FBS at 20 ml/tube and store at 20°C. </p> <p>Add 20 ml chelated FBS per 500 ml bottle of EMEM (final conc’n = 4%). </p> <p><span><strong><em>CaCl<sub>2</sub></em></strong></span></p> <p>Prepare a stock of 0.1 M CaCl<sub>2</sub> in sterile water. Filter sterilize with 0.2μm filter. </p> <p>Add 250μl of 0.1 M CaCl<sub>2</sub> per 500 ml bottle of EMEM (final conc’n = 0.05mM). </p> <p><span><strong><em>Hydrocortisone</em></strong></span></p> <p>Sigma, cat # H0888 Prepare a concentrated stock of 5 mg/ml by dissolving 50 mg hydrocortisone in 10 ml 95% EtOH. Store the concentrated stock at 20°C. </p> <p>Prepare a 0.2 mg/ml stock by adding 0.4 ml of concentrated stock to 9.6 ml of sterile, serum-free EMEM. Aliquot at 1.0 ml/tube; store at 20°C. </p> <p>Add 1.0 ml of 0.2 mg/ml hydrocortisone per 500 ml bottle of EMEM (final conc’n = 0.4μg/ml). </p> <p><span><strong><em>Insulin</em></strong></span></p> <p>Bovine Insulin; Sigma, cat # I6634 Prepare a 5 mg/ml stock: Dissolve 100 mg insulin in 20 ml of 0.005 N HCl (prepared by adding 10μl concentrated HCl to 20 ml water). </p> <p>Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20°C. </p> <p>Add 0.5 ml of 5 mg/ml insulin per 500 ml bottle of EMEM (final conc’n = 5μg/ml). </p> <p><span><strong><em>Epidermal Growth Factor (EGF)</em></strong></span></p> <p>Recombinant human EGF; GibcoBRL, cat # 13247-051 </p> <p>Prepare a 10μg/ml stock: Dissolve 100μg EGF in 1 ml 0.1% BSA (10 mg ultrapure BSA in 10 ml water). Bring volume to 10 ml with 0.1% BSA. Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20°C. </p> <p>Add 0.5 ml of 10μg/ml EGF per 500 ml bottle of EMEM (final conc’n = 10 ng/ml). </p> <p><span><strong><em>Cholera Toxin</em></strong></span></p> <p>ICN, cat # 190329 Prepare a concentrated stock of 10<sup>-5</sup>M by dissolving 1 mg (1 vial) cholera toxin in 1.18 ml distilled water. Store the stock at 4°C (<u>do not freeze the concentrated stock</u>). </p> <p>Prepare a 10<sup>-7</sup>M stock by adding 0.1 ml of the concentrated stock to 10 ml EMEM + 4% FBS. Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20°C. </p> <p>Add 0.5 ml of 10<sup>-7</sup>M cholera toxin per 500 ml bottle of EMEM (final conc’n = 10-10 M). </p> <p><span><strong><em>T3 (3,3’,5-triiodo-L-thyronine)</em></strong></span></p> <span></span><p>Sigma, cat # T6397 </p> <p>Prepare a concentrated stock of 2x10<sup>-4</sup>M by dissolving 13.6 mg T3 in 15 ml 0.02N NaOH, then bringing the volume to 100 ml with PBS. Distribute in 10 ml aliquots and store at 20°C. </p> <p>Prepare a 2x10<sup>-6</sup>M stock by adding 0.1 ml of the concentrated stock to 10 ml PBS. Filter sterilize with 0.2μm filter and aliquot at 0.5 ml/tube; store at 20°C. </p> <p>Add 0.5 ml of 2x10<sup>-6</sup>M T3 per 500 ml bottle of EMEM (final conc'n = 2x10<sup>-9</sup> M). </p> <p> </p> <p><em>Source: OpenWetWare.org</em></p>
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