Cellular senescence is a state where cells can no longer divide, despite the presence of appropriate growth factors. This state can be induced either by telomere shortening (replicative senescence) or by telomere-independent signals, such as deoxyribonucleic acid (DNA) damage and oxidative stress (stress-induced senescence). The state of cellular senescence is associated with the following: irreversible growth arrest; a distinct flat, and enlarged cell morphology; an increase in senescence-associated ß-galactosidase (SA ß-gal) activity; and the expression of cell cycle inhibitors, such as p16INK4a and p21CIP1/WAF1/Sdi1
BioPioneer providesasimple andrapid test for identifying senescent cells and gives science a powerful new tool for examining senescence. This kit is designed to detect SA-beta-galactosidase which catalyzes the hydrolysis of X-gal and produces a blue color in senescence at pH6.0.
Kit Contents:
10X Fixative Solution 15 ml
X-Gal 150 mg
10X Staining Solution 15 ml
100X Staining Solution Supplement A: 1.5ml
100X Staining Solution Supplement B:1.5ml
Materials Supplied by the User
- 1X PBS (Phosphate Buffered Saline):
- N-N-dimethylformamide (DMF)
- 50% glycerol (optional)
- 37°C incubator
- Phase contrast or light microscope
- Polypropylene tubes
Solution Setup
The following protocol is designed for one 35 mm well of a 6-well plate. (1 ml for 35 mm wells/plates, 2.5 ml for 60 mm plates and 5 ml for 100 mm plates).
Polypropulene tubes must be used duringsolution preparation.
1. Prepare a 6ml 1X PBS solution for each 35mm well (not provided).
2. Dilute the 10X Staining and 10X Fixative Solutions with distilled water to make 1X solutions. You will need 930 ul of 1X Staining Solution and 1 ml of 1X Fixative Solution per 35 mm well.
3. Dissolve 20 mg X-gal in 1 ml DMF to prepare a 20X stock solution. Excess X-gal solution can be stored at -20°C in a light resistant container for one month.
Assay Protocol
1. Aspiratethe growth medium from the cells and wash the plate once with 2 ml 1X PBS.
2. Add 1 ml 1X Fixative Solution to the cellsand incubate for 5-10 minutes at room temperature.
3. During the fixingprocess , preparethe Staining Solution.
a. 930 ul Staining Solution
b. 10 ul Staining Supplement A
c. 10 ul Staining Supplement B
d. 50 ul 20 mg/ml X-gal in DMF
4. Remove the fixing solution and wash the fixed cells twice with 2 mL of 1X PBS.
5. Add 1 ml Staining Solution mix to the plate. Incubate 5 hours toovernight at 37°C.
6. Check the cells under a microscope (200 x total magnification) for development of blue color.
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(2)荧光标记法 : 使用二乙酸荧光素(FDP)、碘化丙啶(PI)或异硫氰酸荧光素钠标记的荧光染料与细胞共孵育,用流式细胞仪检测荧光染色阳性细胞的比率。此法其实是(1)法的“荧光”版,但其在灵敏性和准确性方面明显要优于后者。
(3)硝酸镧(La)示踪法: 在正常的生物组织中镧微粒可沉积于细胞间隙,但不能穿过具有1~ 2nm 微小间隙的细胞膜性结构(包括细胞膜和细胞器膜),也不能穿过细胞间的紧密连接。在膜性结构通透性增高时, 镧微粒则可进入细胞、细胞器和紧密连接内, 并在电镜下显示, 镧盐标记技术被认为是一种有效的监测细胞膜通透性变化的标记技术。
(4)LDH释放法: 在正常情况下,细胞内大分子物质LDH 是不能通过细胞膜的, 但在细胞膜受损伤而通透性增加时,可通过受损的细胞膜释放出来。LDH 能较好地反映细胞膜损伤程度。类似的还有检测细胞外K+的漏出率等。
有几个疑问
1:荧光标记到细胞是标记到细胞表面还是细胞质内?
2:荧光应该随着细胞的分化和增殖逐渐消失?是不是分化增殖越快,荧光消失速度越快?
3:有哪些容易操作,成本便宜的荧光物质?
谢谢各位战友
比如你用的CD1a-FITC(如果是鼠单抗IgG1,那对照抗体就要用相同物种的非特异性IgG1-FITC)。注意浓度要相同。一般提供抗体的公司BD,santa cruz等有提供的。其他就按照说明书的推荐浓度和孵育时间。