By Ge Lin, Kristen Martins-Taylor, Ren-He Xu (auth.), Sheng Ding (eds.)
Before the healing power of mobile substitute treatment or the improvement of healing medications for exciting the body’s personal regenerative skill to fix cells broken by means of illness and damage might be absolutely discovered, regulate of stem mobile destiny, immuno-rejection, and constrained cellphone resources has to be conquer. In Cellular Programming and Reprogramming: equipment and Protocols, specialist researchers conceal the newest applied sciences and their comparable mechanisms thinking about the programming and reprogramming of telephone destiny. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, laboratory protocols, and notes to spotlight pointers on troubleshooting and keeping off recognized pitfalls.
Essential and state of the art, Cellular Programming and Reprogramming: tools and Protocols grants to assist scientists trying to strengthen stem cellphone biology so one can greater deal with devastating human illnesses, together with heart problems, neurodegenerative ailment, musculoskeletal affliction, diabetes, and cancer.
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Extra info for Cellular Programming and Reprogramming: Methods and Protocols
2007) New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–9. 6. Brons, I. , Smithers, L. , Trotter, M. , Chuva de Sousa Lopes, S. , Howlett, S. , Pedersen, R. , and Vallier, L. (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–5. 7. Kaufman, M. H. (1992) The Atlas of Mouse Development, Elsevier Academic Press, London. 8. Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.
Divide the cell suspension equally among the flasks and incubate in a Tissue Culture Incubator. This is the primary plating and is denoted as passage 0. Change the medium the next morning to remove excess debris. 2. Passage 0 MEFs require passaging when they are nearly confluent. This is initially 1–3 days after the primary plating and depends on the initial plating density and survival (see Note 13). Cells are passaged 1:4 (each passage 0 flask is split into 4). To passage, remove medium from each T175 flask, rinse with 5 ml PBS, add 3 ml MEF Passaging Medium, and incubate for 5 min at 37°C.
Add 75 ml of Dual-Glo Luciferase Reagent and mix by pipetting three times. Transfer all 150 ml of each sample to separate wells of a 96-well white microplate. Protect the plate from light and incubate at room temperature for 10 min. Measure the firefly luminescence using a luminometer to measure total luminescence for each sample for 1 s (see Note 38). This first measurement detects the firefly luciferase expressed from the Oct3/4 plasmids. After the firefly luminescence is recorded, to each well add 75 ml of the Dual-Glo Stop & Glo reagent that has been prepared according to the manufacturer’s protocol.