By Kursad Turksen
A set of state-of-the-art how to learn and manage epidermal cellprecursors and mature epidermal cells. those protocols hide assorted equipment and versions for culturing epidermal cells, for enriching very early epidermal progenitors, and for learning epidermal mobilephone dedication and differentiation either in vitro and in vivo. issues of unique curiosity comprise the derivation, characterization, and software of epidermal stem cells, mature epidermal cells and their characterization, and functions in regenerative medication. those with ease reproducible thoughts increase our knowing of the biology of epidermal cells and in their application in general tissue homeostasis and regenerative drugs functions.
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Extra info for Epidermal cells: methods and protocols (Volume 289)
2. Subculture Keratinocytes should be subcultured before extensive differentiation or growth arrest (~60–80% confluence). If intending immediate subculture, handle cells quickly at ambient temperature. Otherwise, keep cell suspension at 4°C to maintain viability. 1. Rinse cultures thoroughly with PBS-AF twice. 05% EDTA to cover the entire surface and incubate at 37°C for 10–15 min to initiate cell detachment. 2. When intercellular spaces look enlarged, remove EDTA and add prewarmed trypsin–EDTA to cover.
Clone the selected colonies using the cloning cylinder. 3. Detection of the Ectopic Protein (Shh or Wnt-3) Expression by Western Blotting 1. 2. to confluence in 60-mm culture dishes (see Note 3). 2. Wash the culture twice with ice-cold PBS. 2 mL of the extraction buffer, scrape the cells with the rubber policeman, and mix well. 5-mL microtube, centrifuge (10,000 rpm, 5 min), and collect the supernatant. 3. Determine the quantity of protein in the extract using the protein assay kit with protein standard.
27 subsequent analysis. 2 % (w/v) fatty-acid-free bovine serum albumin. Maintain cells in suspension for desired time intervals up to 72 h. If longer incubation times are required, culture media need to be replaced by centrifugation of the cells, resuspension in fresh medium, and reseeding on agarose. When removing aliquots of cells for analysis, mix the culture by gentle pipetting with a serological pipet, then remove the desired volume and analyze cells. 5–5 × 104 cells) and seed into 24-well plates in optimal growth medium containing all supplements.
Epidermal cells: methods and protocols (Volume 289) by Kursad Turksen