小中大
以下是SA-β-Gal染色法的最经典文献.
1.a biomarker that identifies senescent human cells in culture and in aging skin in vivo
Proc.Natl.Acad.Sci.USA Vol:92, pp:9363-9367,1995.
2.Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cells. Oncogene. 2006 Jan 12;25(2):176-85
方法:
SA-B-gal staining
At 10 days after the addition of Sirtinol or Splitomicin to the culture media, the cells were washed twice with phosphate-buffered saline (PBS) and then fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 10 min. The cells were then incubated at 37ºC (NO CO2)for 10 hr with staining solution (40 mM citric acid sodium phosphate, pH 6.0, 1 mg/ml 5-bromo-4-chloro-3-isolyl-B-D-galactoside [X-gal, Fisher, Pittsburgh, PA], 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2). After being washed twice with PBS, the cells were incubated with 4’, 6-diamidino-2-phenlindole dihydrochloride (DAPI, 1 ug/ml, DOJINDO, Tokyo, Japan) at 4ºC for 2 hr. SA-B-gal-positive cells were enumerated by counting over 400 cells in three independent fields.
我也刚开始这个试验
请问
1. 40 mM citric acid sodium phosphate 是不是用柠檬酸来把磷酸钠两者混合调成, 另外这里的PH6.0是不是指整个staining solution的PH,用什么来调最为合适??
2.不知如何做到在无二氧化碳的环境，请大虾们指点一下??
3.DAPI只是染核以便计数细胞而已,是否可以用其它染料取代,比如核固红把核染成红色??
4.有谁用过碧云天的这种试剂合,效果如何??
