小鼠胚胎,干细胞,体外TE分化 z-bo 2022-02-05 492

　　Abstract: Embryonic stem cells have the characteristics of immortal proliferation, self-renewal and multidirectional differentiation in vitro, and can be induced to differentiate into almost all cell types in vivo. This method mainly introduces the in vitro EB differentiation process of mouse embryonic stem cells.

　　Experiment Overview

　　This method mainly introduces the in vitro EB differentiation and identification process of mouse embryonic stem cells.

　　Main reagent

　　High-sugar DMEM (Gibco, product number: 12800-017 powder), DMEM/F12 (Gibco, product number: 12500-062 powder), fetal bovine serum (FBS) (Gibco, product number: 16000044) non-essential amino acids (NEAA) (Gibco, Product number: 11140050), 0.25% (1X) pancreatin containing EDTA4Na, liquid (Gibco, product number: 25200072), gelatin (BD, product number: 354277), penicillin (SP) (Gibco, product number). : 15140122), L-glutamine (Gibco, item number: 25030081), serum substitute (Gibco, item number: 10829028) 10% FBS culture medium (Gibco, item number 10099141) Main equipment 1 super cleaning station

　　2, carbon dioxide incubator (Thermo, catNO.3131)

　　3, 6cm petri dish (corning, catNO.430166), 10cm petri dish (corning, catNO.430167)

　　4, 15ml centrifuge tube (corning, catNO.430791), 50 ml centrifuge tube (Corning, article number 430829), cryopreservation tube (Corning, article number 430659)

　　5. Upright microscope

　　Experimental steps

　　①Conventional cell digestion method, only need to put the cell cluster into the straw with a rough mouth pipette;

　　②Transfer the cell mass to a 6 cm or 10 cm petri dish and incubate it with 10% FBS medium (add 5 ml of medium to each 6 cm petri dish).

　　③Place the petri dish in a carbon dioxide incubator at 37°C for culture, then take it out once or twice a day, gently shake with a rough pipette or suspend the cells with a pipette, separate the clusters, and observe daily;

　　④ After 3 days of normal culture, the cell clusters began to stratify. The outer layer is a large layer of endoderm-like cells, while the inner layer of cells is small and compact. These are ectoderm-like cells and stem cells. These are simple embryonic cells. Incubate for 5-10 days. The layers increase and cavities appear, and the whole embryo becomes round vesicles, blastocysts.

　　⑤ Collect blastocysts, extract mRNA, reverse transcription into cDNA, use 3 germ layer primers for PCR, detect the expression of 3 germ layer genes, and determine whether there are 3 differentiated bacterial layers. Whether the cell has the ability to differentiate into three germ layer cells in vitro.