Method for the prophylaxis and/or treatment of medical disorders
a medical disorder and prophylaxis technology, applied in the field of medical disorders, can solve the problems of affecting a significant proportion of the population, and not being completely effective, and being free of adverse side effects
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example 1
[0118] The differentiated human keratinocyte cell line, HaCaT [9] was used in the in vitro assay. Cells at passage numbers 33 to 36 were maintained as monolayer cultures in 5% V / V CO2 at 37° C. in Keratinocyte-SFM (Gibco) containing EGF and bovine pituitary extract as supplied. Media containing foetal calf serum were avoided because of the high content of IGF-I binding proteins in serum.
[0119] Feeder layer plates of lethally irradiated 3T3 fibroblasts were prepared exactly as described by Rheinwald and Green [10].
example 2
[0120] Cells were grown to 4 days post confluence in 2 cm2 wells with daily medium changes of Keratinocyte-SFM, then the medium was changed to DMEM (Cytosystems, Australia), with the following additions: 25 mM Hepes, 0.19% w / v, sodium bicarbonate, 0.03% w / v glutamine (Sigma Chemical Co, USA), 50IU / ml penicillin and 50 μg / ml streptomycin (Flow Laboratories). After 24 hours, IGF-I or tIGF-I was added to triplicate wells, at the concentrations indicated, in 0.5 ml fresh DMEM containing 0.02% v / v bovine serum albumin (Sigma molecular biology grade) and incubated for a further 21 hours. [3H]-Thymidine (0.1 μCi / well) was then added and the cells incubated for a further 3 hours. The medium was then aspirated and the cells washed once with ice-cold PBS and twice with ice-cold 10% v / v TCA. The TCA-precipitated monolayers were then solubilized with 0.25M NaOH (200 μl / well), transferred to scintillation vials and radioactivity determined by liquid scintillation counting (Pharmacia Wallac 1410 ...
example 3
[0121] HaCaT conditioned medium (250 μl) was concentrated by adding 750 μl cold ethanol, incubating at −20° C. for 2 hours and centrifuging at 16,000 g for 20 min at 4° C. The resulting pellet was air dried, resuspended thoroughly in non-reducing Laemmli sample buffer, heated to 90° C. for 5 minutes and separated on 12% w / v SDS-PAGE according to the method of Laemmli (1970).
[0122] Separated proteins were electrophoretically transferred to nitrocellulose membrane (0.45 mm, Schleicher and Schuell, Dassel, Germany) in a buffer containing 25 mM Tris, 192 mM glycine and 20% v / v methanol. IGFBPs were then visualised by the procedure of Hossenlopp et al [11], using [125I]-IGF-I, followed by autoradiography. Autoradiographs were scanned in a BioRad Model GS-670 Imaging Densitometer and band densities were determined using the Molecular Analyst program.
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