Though minimal Ca2+, serum-totally free problems have been broadly utilized to lifestyle basal proliferating cells, with regards to literature, differentiation could be induced with numerous other procedures. In truth, a single can use a Ca2+ [three] or serum/Ca2+ switch [6,seven], or cell confluence [8].RU-19110 structure It has thus turn into straightforward to get misplaced in this jungle of methods and to formulate improper interpretations. Ca2+ has been demonstrated to be a big aspect in managing keratinocyte differentiation [nine]. It can from time to time be read here and there that the Ca2+ gradient will increase linearly from the basal to the corneocyte layer, but about the past 30 many years, several groups have demonstrated with distinct approaches that Ca2+ is distributed as a non-linear gradient in human skin with a very low concentration in the basal layer, the cheapest focus currently being in the supra-basal / early spinosum levels and with a marked boost in the late spinosum, granular and corneosum compartments [102]. In current a long time, a simplified consensus has appeared such as to the use of reduced Ca2+ (<0.1mM) supplements for proliferating and high Ca2+ (> 1mM) dietary supplements for inducing differentiation. Strikingly, an unique report shown that ideal rate of keratinocyte proliferation was attained at about .3 mM Ca2+, but dropped at decreased Ca2+ concentrations (<0.1mM Ca2+) in serum free conditions [3], thereby coinciding with the endogenous gradient of Ca2+ in human epidermis. The induction of keratinocyte differentiation is even more confusing. Indeed, authors have described a permissive effect of high Ca2+ on differentiation marker expression [7,13], whilst others have shown a high-Ca2+ mediated induction of differentiation markers [14,15]. Serum has been reported to trigger proliferation arrest and to induce the differentiation of mouse primary keratinocytes [2,6,16]. Besides, a recent study showed that culturing human keratinocytes in serum-free condition for several passages made them unable to form stratified layers in reconstituted skin model [17]. Paradoxically, it has been suggested the vitamin A contained in serum did counteract differentiation of keratinocytes [18] and to repress keratin 10 expression. Consequently, serum-free media were developed and the BPE supplementation has become a standard in serum-free media, since it increases basal keratinocyte proliferation and increases their survival. It may also be noted that BPE inhibits terminal differentiation partially [3]. Furthermore, it is accepted that the differentiation of granular-like keratinocytes is achieved in confluent, multilayer keratinocyte cultures [8,14,15], whiles others have demonstrated that terminal differentiation could be achieved in detached isolated keratinocytes [7]. Finally, it has been shown that culture at the air-liquid interface is required for cornified barrier formation in orthotypic cultures [19]. A further interesting feature of human skin is its exposure to large variations in temperature. At a comfortable external temperature (about 30), average physiological human skin reaches about 32 [20]. Surprisingly, almost all the studies conducted on cultured keratinocytes have been performed at 37. To our knowledge, only one group studied the effect of incubation at 32 on the proliferation/differentiation balance of keratinocytes. Indeed, Ponec et al demonstrated that the deepidermized dermis regenerated a well-differentiated epidermis with a morphology showing a higher resemblance to the native epidermis than the cultures grown at 37 [21,22]. In addition, it has recently been reported that 31 was optimal for ATP release by keratinocytes [23]. Because, ATP has been thought to be a potent autocrine factor in the regulation of the proliferation/differentiation balance [24], lower temperature may optimize differentiation of keratinocytes. Quantifying shift in the rate of differentiation of a cell population implies a prior characterization and quantification of keratinocyte phenotypes (basal, spinal-like, granular-like, corneocytes), defined with the use of molecular markers. Consequently the qualitative and quantitative expression of these molecular markers has to be determined in the cell models and compared to the ones of in vivo skin. However, this very equivalence has rarely been taken into consideration and has probably led to over-interpretations considering that single protein induction may be extrapolated as an increase of the differentiation rate. Therefore, in this study, we developed a set of differentiation markers beyond those classic secondary filament markers, in order to discriminate between single gene induction and the wide rearrangement of gene/protein expression expected to occur during differentiation. We indeed highlighted the limits of qPCR and immunoblotting (IB), but also showed the advantages of making parallel analyses with immunocytofluorescence (ICF) and flow cytometry (FC), when studying variations in the differentiation rate. Regarding these technical issues, we studied the paradoxical connection between calcium, serum, multilayer culture, and incubation temperature on the differentiation of in vitro keratinocytes. Unlike some former reports, we demonstrated that calcium switch is indeed a potent model for inducing calciumdependent genes, but is not an appropriate procedure for assessing keratinocyte differentiation. In addition, we demonstrated that a synergic stimulation by calcium, serum, confluence and a lower incubation temperature accelerated the differentiation rate.HaCaT cell line was grown in Dulbecco's minimal essential medium, DMEM, (Gibco) supplemented with 2% fetal calf serum (FCS), Kanamycin (100 /ml), Sodium Pyruvate (1mM) and Ca2+ was adjusted to 0.2 mM for basal cell culture. Ca2+ was adjusted to a final 1.8 mM to induce differentiation. hNEK cells were obtained from Lifescience Inc. and plated in 10 cm round dishes in basal KSF-SFM medium at a density of 250 k cells/dish. Basal KSF-SFM medium was supplemented with bovine pituitary extract, EGF, glutamine and kanamycin as advised by the manufacturer (Invitrogen). 2% FCS or Ca2+ adjusted to 1.8 mM were added to basal KSF-SFM media to induce keratinocyte differentiation.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the French National Institute for Medical Research (INSERM). The Committee on the Ethics approved the protocol for Animal Experiments of University of Lille 2 (Permit number: CEEA 08/2009). All efforts were made to minimize suffering. After sacrifice, the backskins of C57BL/6J mice were shaved with an electric razor prior to skin removal. The isolation and culture protocol for epithelial cells was derived from Nowak and Fuchs [25]. The skin was set hair side up on the dissecting pad. Using the scalpel, the fat and blood vessels covering the dermis were scraped until the dermis was clearly and uniformly exposed. Then the skin, dermis side down, was put in solution with 0.25% trypsin, making sure that the skin was freely floating with an unsubmerged epidermis. The skin was incubated overnight at 4. The following day, using a scalpel and forceps we separated the epidermis from the dermis by scraping along the top to remove the epidermis. The dermis was removed from the dish and the largest pieces of epidermal tissue were briefly minced. After pipetting up and down to triturate the tissue, Defined-keratinocyte medium (Gibco) containing 10% FCS was added to inactivate the trypsin. The epidermis mixture was then passed through a 70 cell strainer. After washing the strainer with 5 ml of medium, the cell suspension was centrifuged for 10 min at 250 g. Epidermal cells were then re-suspended in fresh Definedkeratinocyte medium without serum and grown in adapted petri dishes. To avoid fibroblast development, cells were cultured in this medium for 5 days. They then were grown in basal KSFM medium to increase cell growth.After total mRNA extraction and purification with TRI REAGENT(Sigma-Aldrich), mRNA were subjected to DNAse treatment (Ambion) at 0.25祃 DNAse per of RNA for 25 min at 25. Afterwards, 10 mRNA were purified in a V/V phenol/chloroform/AIA solution (Fluka) with 5% Sodium Acetate 3M. The upper phase was supplemented with 10% Sodium Acetate 3M and 2.5 V 100% Ethanol and kept at -20 overnight in order to precipitate. After a brief wash in 70% Ethanol, pellets were left to dry and then re-suspended in 30 water. After an agarose gel check of mRNA quality, 2 of mRNA were subjected to reverse transcription as reported elsewhere [26]. Real-time quantitative PCR was performed on a Cfx C1000 system (Biorad). For each reaction, 12.5 ng of cDNA was placed in a final reaction mixture of 15 containing 7.5祃 of 2x SsoFastTM EvaGreenSupermix (Biorad) and 200nM primer pairs (see Table 1). The PCR protocol was: an initial 30 sec denaturation step at 95, and 40 cycles of [4 sec at 95, 30 sec at 60] and a final dissociation curve to control the specificity of the amplification. The housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to normalize variations in RNA extractions, the degree of RNA degradation and variability in RT efficiency. GAPDH mRNA has been selected as the most invariant gene in a set of 6 reporters: HPRT mRNA, GAPDH mRNA, 18S rRNA, ATF6 mRNA, DNAJ mRNA and KRT17 mRNA. Mean Ct were calculated for the four experimental procedures of cell culturing: (-Ca2+, -FCS), (+Ca2+, -FCS), (-Ca2+, +FCS) and (+Ca2+, +FCS) and Variances of mean Ct were figured out for each mRNA in order to determine the invariant. Variances of 18S rRNA, HPRT mRNA, GAPDH mRNA, ATF6 mRNA, DNAJ mRNA and KRT17 mRNA were respectively: 2.57, 0.1, 0.25, 0.22, 0.46 and 0.68. We used the comparative Ct method to quantify mRNA levels.An ice-cold buffer (pH 7.2) containing 10 mM PO4Na2/K buffer, 150 mM NaCl, 1 g/100 ml sodium deoxycholate, 1% Triton X-100, 1% NP40, a mixture of protease inhibitors(Sigma-Aldrich), and a phosphatase inhibitor (sodium orthovanadate Sigma-Aldrich) was applied to previously PBSwashed cells in dishes. After 30min incubation on ice, the protein extract was transferred to 1.5 ml tubes and subjected to sonication. After 10 minutes of centrifugation at 15,000 g, the pellet was transferred into a clean tube prior to a determination of the protein concentration using a BCA Protein Assay (Pierce). 25 of total protein were loaded onto a 10% polyacrylamide gel before an SDS-page was performed. After electrophoresis, proteins were transferred to a nitrocellulose membrane using a semi-dry electroblotter (Bio-Rad). The membrane was blocked in a TNT +5% (W/V) milk (15 mM Tris buffer, pH 8, 140 mM NaCl, 0.05% Tween 20, and 5% non-fat dried milk) for 30 min at room temperature, then soaked in primary antibody diluted in TNT +1% milk for either 2 h at room temperature or overnight at +4. After three washes in TNT, the membrane was soaked in secondary antibody diluted in TNT+1% milk for 1h at room temperature. The membrane was processed for chemiluminescence detection using Luminata Forte Western HRP Substrate (Millipore) according to the manufacturer's instructions. After a 10 min bath in Re-blot PLus Mild SOlution (Millipore), membrane was blotted again. The primary antibodies used were: anti-Keratin 5 (Covance) at 1/2000, anti-Keratin 10 (Covance) at 1/1000, anti-Involucrin (Sigma) at 1/500, anti-Filaggrin (Abcam) at 1/200, anti-Vimentin (Santa Cruz) at 1/400, anti-GAPDH (Santa Cruz) at 1/250. Secondary antibodies were coupled to either green fluorochrome DyeLight-488 (Molecular probes) or red fluorochrome AlexaFluor-546 (Molecular probes).2872801 Nuclei were counterstained with Dapi (1/400, Dako)1/2000, anti-Keratin 10 (Covance) at 1/100 and anti-Keratin 10 (Abcam) at 1/200, anti-Involucrin (Sigma) at 1/1000 and antiInvolucrin (Abcam) at 1/400, anti-Filaggrin (Abcam) at 1/800, anti-Loricrin (Abcam) at 1/1000.Flow cytometry was performed with a CyAnTM ADP Analyser. Cells were harvested, split into 1 million cell samples in 15 ml tubes before fixation with 1ml of 70% Ethanol at -20 overnight. Cells were washed twice with PBS / 4% BSA / 0,1% TritonX100 and finally incubated at RT for 30 min. Primary antibodies were diluted in 100 祃 PBS-BT at the same dilution as that for immunofluorescence and incubated with cells at RT for 1h. After a first quick wash in PBS, a second wash was done at RT for 30 min. Secondary antibodies: anti-rabbit IgG coupled to DL-488 and anti-mouse IgG coupled to AF-647 were diluted respectively at 1/2000 and 1/4000 and incubated with cells at RT for 30 min. Afterwards, cells were washed out 3 times at RT for a total of 30 minutes. The flow cytometer was calibrated with rainbow beads before each experiment. 488 and 642 lasers were used. Data were analyzed with FlowJo software (v 8.7).Confocal imaging experiments were performed using a confocal laser scanning microscope (LSM 780 Carl Zeiss MicroImaging, Inc) and a plan-Apochromat 401.3 NA oil immersion objective.