A method of cell induction
A cell and cell body technology, applied in animal cells, cell culture medium, vertebrate cells, etc., can solve the problem of high risk of clinical application, and achieve the effect of simple operation and good repeatability
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Embodiment 1
[0098] Example 1 Transformation from fibroblasts to adipocytes
[0099] Taking a 12-well plate as an example (corning, 3335), prepare each well with 20 μg / ml Matrigel solution (BD, 354277) 1×DMEM, coat for 12 hours, remove the coating solution and wash once with 1×PBS flushing solution.
[0100] Mouse embryonic fibroblasts (C57, prepared at E13.5) or adult foreskin fibroblasts (HFF20y, Beijing Stem Cell Bank) were evenly seeded in each well, 1 × 10 per well 4 Each cell was cultured with basal medium (high glucose DMEM (Gibco, C12430500BT), double antibody) plus 10% fetal bovine serum (Gibco, 16000-044) for 24 hours. Remove the culture medium and wash again with PBS.
[0101] The above-mentioned fibroblasts were added to fat induction medium: (N2B27 medium: DMEM / F12 (Gibco, 10565018) and Neurobasal (Gibco, 21103-049) were mixed in a ratio of 1, N2 additive (100×, Gibco , 17502048), B27 additive (50×, Gibco, 17504044), 2% bovine serum albumin (1000×, sigma, A8022), β-mercaptoe...
Embodiment 2
[0106] Example 2: Transformation from fibroblasts to immortalized cells
[0107] Taking a 12-well plate as an example (corning, 3335), each well was prepared with 20 μg / mL matrigel solution (BD, 354277) 1×DMEM, coated for 12 hours, and washed once with 1×PBS after removing the coating solution.
[0108] Mouse embryonic fibroblasts (prepared from C57, E13.5) or tail tip fibroblasts (prepared from one week after birth or from adult mice) were evenly seeded in each well, 2×10 per well 4 Each cell was cultured with basal medium (high glucose DMEM (Gibco, C12430500BT), double antibody) plus 10% fetal bovine serum (Gibco, 16000-044) for 24 hours. Remove the culture medium and wash again with PBS.
[0109] The above-mentioned fibroblasts were added to the immortalization induction medium: (N2B27 medium: DMEM / F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) were mixed at a ratio of 1, and N2 additive (100×, Gibco, 17502048), B27 additive (50×, Gibco, 17504044), 2% bovine seru...
Embodiment 3
[0114] Example 3: Detection of Proliferation and Senescence Genes
[0115] The experimental method is as follows: Immortalized cells SMPC (mouse embryonic fibroblasts as control) were collected. (a) RNA was extracted by kit method. Add an appropriate amount of TRIzol to the cell pellet to lyse the cells, add 1 / 5 volume of chloroform, vortex and mix well, let stand on ice for 3 minutes, centrifuge at 10000g, 4°C for 15 minutes. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of 75% ethanol and transfer it to the adsorption column, centrifuge at 10,000 g for 15 seconds, discard the liquid in the collection tube, wash with Wash Buffer I once, and add 10 μL DNase I+70 μL Buffer RDD, incubate at room temperature for 15 minutes to digest DNA, then wash once with 350 μL Wash Buffer I, once with 500 μL Wash Buffer II, and elute RNA with RNase-Free Water. Concentrations were determined with a UV / Vis spectrophotometer. (b) Reverse transcription of RNA i...
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