Skin Keratinocyte Culture on Dermal Matrix

Pre-confluent keratinocyte cultures (km) were disaggregated and transfected with SMART pool siRNAs or non-targeting control siRNAs, or with lentiviral shRNAs. 24 hr post-siRNA transfection or >7 days post-shRNA infection, keratinocytes were collected and reseeded on irradiated de-epidermised human dermis (Sen et al., 2010 (link)) in six-well Transwell plates with feeders and cultured at the air–liquid interface for 3 weeks. The cultures were then fixed in 10% neutral buffered formalin (overnight), paraffin embedded and sectioned for histological staining. 6 μm thick sections were labelled with haematoxylin and eosin or appropriate antibodies. H and E stained images were acquired with a Hamamatsu slide scanner and analysed using NanoZoomer software (Hamamatsu).

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Mishra A., Oulès B., Pisco A.O., Ly T., Liakath-Ali K., Walko G., Viswanathan P., Tihy M., Nijjher J., Dunn S.J., Lamond A.I, & Watt F.M. (2017). A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis. eLife, 6, e27356.

Publication 2017

slide scanner NanoZoomer software

Antibodies Dermis Eosin Formalin Haematoxylin Human Infection Keratinocytes Paraffin Shrna Sirna Transfection

Corresponding Organization : King's College London

Other organizations : University of Cambridge, University of Dundee, Wellcome Centre for Cell Biology, University of Edinburgh, Brain Physiology Lab, Université Paris Cité, Microsoft Research (United Kingdom), Wellcome/MRC Cambridge Stem Cell Institute, Medical Research Council

Top 5 similar protocols

Variable analysis

independent variables

SiRNA treatments (SMART pool siRNAs or non-targeting control siRNAs)
Lentiviral shRNA treatments

dependent variables

Histological changes in keratinocyte cultures after 3 weeks of air-liquid interface culture on de-epidermised human dermis

control variables

Pre-confluent keratinocyte cultures (km)
Irradiated de-epidermised human dermis substrate
Culture conditions (six-well Transwell plates with feeders, 3 weeks of air-liquid interface culture)
Fixation and processing (10% neutral buffered formalin, paraffin embedding, sectioning)
Histological staining (haematoxylin and eosin, antibody labeling)
Imaging (Hamamatsu slide scanner, NanoZoomer software)

controls

Non-targeting control siRNAs

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