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9860
Senescence β-Galactosidase Staining Kit
细胞检测试剂盒
检测试剂盒

Senescence β-Galactosidase Staining Kit #9860

引用 (63)
FUNC Image 1 - Senescence β-Galactosidase Staining Kit

pH 值 为 6 时,对细胞群倍增数为 29(左图)的正常 WI38 细胞以及细胞群倍增数为 36(右图)的衰老 WI38 细胞进行 β-半乳糖苷酶染色。

FUNC Image 2 - Senescence β-Galactosidase Staining Kit

pH 为 6.0 时,对未处理的 MCF-7 细胞(左图)和经依托泊苷 #2200(12.5 μM,24 小时)处理并恢复 4 天的衰老 MCF-7 细胞(右图)进行 β-半乳糖苷酶染色。

购买 # 9860S
产品货号 规格 价格 库存
9860S
1 个试剂盒(125 个 35 mm 孔)

产品包括 数量(计数)
10X Fixative Solution 1 x 15 ml
10X Staining Solution 1 x 15 ml
100X Solution A 1 x 1.5 ml
100X Solution B 1 x 1.5 ml
X-Gal 1 x 150 mg

实验步骤

打印

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9860 Senescence β-Galactosidase Cell Staining Protocol

A. 溶液和试剂

Reagents provided are sufficient to stain 125 x 35 mm wells.

提供的试剂

  1. 10X Fixative Solution #11674
  2. 10X Staining Solution #11675
  3. 100X Solution A #11676
  4. 100X Solution B #11677
  5. X-Gal #11678

其他试剂(未提供)

  1. 1X PBS
  2. DMF (Dimethylformamide) #12767
  3. Polypropylene tubes
  4. 37°C dry incubator (No CO2)
  5. Phase contrast or light microscope
  6. 70% glycerol (optional)
  7. DMSO (Dimethyl Sulfoxide) #12611

B. Solution Preparation

NOTE: All solutions should be prepared just prior to use.

Volumes are for one 35 mm well of a 6-well plate. Volumes in the procedure should be approximately half that of the tissue culture media. (e.g. 1 ml for 35 mm well/plates containing 2 ml of media, 2.5 ml for 60 mm plates containing 5 ml of media, and 5 ml for 100 mm plates containing 10 ml of media).

  1. PBS: Prepare at least 6 ml 1X PBS per 35 mm well
  2. Fixative Solution: Dilute the 10X Fixative Solution to a 1X solution with distilled water. You will need 1 ml of the 1X solution per 35 mm well.
  3. Staining Solution: Redissolve the 10X Staining Solution by heating to 37°C with agitation. Dilute the 10X staining solution to a 1X solution with distilled water. You will need 930 ul of the 1X Staining Solution per 35 mm well.
  4. X-Gal:

    IMPORTANT: Always use polypropylene plastic or glass to make and store X-gal. Do not use polystyrene.

    Dissolve 20 mg of X-gal in 1 ml DMF to prepare a 20 mg/ml stock solution. Excess X-gal solution can be stored in -20°C in a light resistant container for up to one month.

    Note: DMSO can be used in place of DMF.

  5. β-Galactosidase Staining Solution: For each 35 mm well to be stained, combine the following in a polypropylene container:
    1. 930 µl 1X Staining Solution (See step 3)
    2. 10 µl 100X Solution A
    3. 10 µl 100X Solution B
    4. 50 µl 20mg/ml X-gal stock solution (see Step 4)

    IMPORTANT: Due to variations in water pH, please be sure that the β-Galactosidase Staining Solution has a final pH of 6.0 (A pH 5.9-6.1 is acceptable). pH differences can affect staining: A low pH can result in false positives and high pH can result in false negatives. If necessary, use HCl or NaOH to lower or raise pH, respectively.

C. Procedure

  1. Remove growth media from the cells.
  2. Rinse the plate one time with 1X PBS (2 ml or a 35 mm well plate, or match volume of media)
  3. Add 1 ml of 1X Fixative Solution to each 35 mm well. Allow cells to fix for 10-15 min at room temperature.
  4. Rinse the plate two times with 1X PBS (SAFE STOP) Alternatively, plates can be left in 1X PBS, covered at +4°C overnight.
  5. Add 1 ml of the β-Galactosidase Staining Solution to each 35 mm well (see Solution Preparation Step 5).
  6. Important: Seal plate with parafilm to prevent evaporation. Evaporation can cause crystals to form.

  7. Incubate the plate at 37°C at least overnight in a dry incubator (no CO2).
  8. Note: The presence of CO2 can cause changes to the pH which may affect staining results.

  9. While the β-galactosidase is still on the plate, check the cells under a microscope (200X total magnification) for the development of blue color.
  10. For long-term storage of the plates, remove the β-Galactosidase staining solution and overlay the cells with 70% glycerol. 储存在 4°C。

发布​于 2020 年 8 月 

实验步骤编号:2164

产品说明

Senescence β-Galactosidase Staining Kit 便捷地提供了 pH 为 6 时检测 β-半乳糖苷酶活性所需的试剂,这种活性是衰老细胞的一种已知特性。对细胞和冷冻组织使用该试剂盒的情况已有论文发表。该试剂盒包含进行该检测所需的所有试剂。

背景

复制能力有限是多数正常细胞的一个典型特征,在衰老时达到顶点,细胞处于仍然有活力的停滞状态 (1)。培养物中的血清或传代不会刺激衰老细胞发生分裂,并且衰老会产生特殊的细胞周期特点,此特点有别于多数损伤诱导的停滞进程或接触抑制 (2)。细胞增大、pH 依赖性 β-半乳糖苷酶活性表达 (3) 以及基因表达模式改变 (4,5) 是衰老细胞的进一步特征。

  1. Goldstein, S. (1990) Science 249, 1129-1133.
  2. Sherwood, S. W. et al. (1988) Proc. Natl. Acad. Sci. , USA 85, 9086-9090.
  3. Dimri, G. et al. (1995) Proc. Natl. Acad. Sci., USA 92, 9363-9367.
  4. Cristofalo, V. J. et al. (1998) Crit. Rev. Eukaryot Gene Expr. 8, 43-80.
  5. Linskens, M. H. et al. (1995) Nucleic Acid Res. 23, 3244-3251.

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产品引用:63

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