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G1073-100T Aging Cell β-galactosidase Staining Kit

Specifiction: 100 T
Product use:Staining of senescent tissue or senescent cells.
Storage and transportation: 

The X-Gal powder was dissolved with 5ml DMF in the kit, divided into 1ml/ tube, and stored at -20 in a dark way to avoid repeated freeze-thaw. After the solution was prepared, the shelf life of X-Gal was 3 months.If it continues to be used beyond the expiration date, it may affect the experimental results.

The kit is valid for one year.

T:
  • G1073-100T
  • Servicebio

Product Description


Product Description

Most normal cells have a limited ability to divide, and when they cannot divide, they become senescent.Senescent cells are usually larger and have high β-galactosidase expression, and higher β-galactosidase activity can be detected at pH6.0.The kit is based on this principle, aiming at the aging related β-galactoidase activity level up-regulation and aging tissue or cell staining kit.X-Gal was used as substrate to produce blue products catalyzed by senescent cell-specific β-galactosidase and observed under light microscope.

Product Usage
Product Name

Code

Specifiction

β-galactosidase staining fixation solution

G1073-1

100 ml

β-galactosidase staining solution A

G1073-2

100 ml

β-galactosidase stain B

G1073-3

1.25 ml

DMF

G1073-4

5 ml

X-Gal

G1073-5

100 mg, powder,1 tube


Usage:


1.  Reagen preparation

(1) Preparing your own PBS buffer solution.

(2) X-Gal powder 100mg was thoroughly dissolved and mixed with 5mL DMF to obtain X-Gal solution, which was then divided into 1.5mL centrifuge tubes with 1mL for each tube and stored at -20℃ away from light.

(3) The dyeing solution was prepared in the proportions shown in the table below:


β-galactosidase staining solution A

940 μl

β-galactosidase stain B

10 μl

X - Gal solution

50 μl


2. Staining step

(1) For adherent cell

a) Cells cultured in 6-well plates (or cell slides) were sucked out of cell culture medium, washed twice with PBS, then 1ml β-galactosidase staining fixation solution was added, and fixed at room temperature for 15min.

b) The fixed solution was discarded and the cells were washed with PBS for 3 times, 2min each

c) PBS was sucked out as far as possible, 1ml of staining solution was added to each well, and incubated at 37℃ for 2h until overnight.Note: Do not incubate in 37℃ carbon dioxide incubator.During staining, the color development should be observed in time. If the expression of β-galactosidase in the sample is high, the staining can be completed within a few hours.If the expression of β-galactoside enzyme is low, the incubation time should be extended appropriately, during which the 6-well plate should be sealed with plastic wrap or Parafilm to prevent the evaporation of the liquid.

d) Observation under ordinary light microscope.If it is not possible to observe and count in time, the staining working fluid can be removed, the cells can be washed with PBS, and then 2ml PBS can be added and stored for 1 week at 4℃.Or, after removing the staining fluid, add 70% glycerin to cover the cells, and the cells can be stored for a long time at 4℃.If it is a cell slide, it can be dehydrated by gradient ethanol and then drip sealed tablet made into permanent slice for preservation.

(2) For frozen section

a) The frozen sections were balanced at room temperature for 10min.Circle the tissue with a composition stroke.

b) An appropriate amount of β-galactosidase staining fixation solution was added to the tissue to completely cover the tissue and fixed at room temperature for 20min.

c) The tissue sections were soaked and washed by PBS for 3 times, 5min each.

d) The section is placed in a light-proof wet box, and an appropriate amount of staining solution is added to the tissue to completely cover the tissue.The wet box was incubated at 37℃, and the color rendering was observed under the microscope every 2h. If no color rendering was observed, the incubation was continued until the senile cells showed color on the tissue.If the sample is to be incubated overnight, a sufficient amount of the dyeing solution should be added to prevent the liquid from evaporating.

e) After the tissue color was developed, the staining solution was removed, and the sections were soaked in PBS for two times and soaked in pure water for two times

f) Drops of nuclear solid red dye were added for 3min and washed for 3 times.This step is not required.If the nucleus needs to be stained, we can use our nuclear fixation red dye (G1035).

  g) Dehydrate with anhydrous ethanol for 2 times, 5min each, xylene transparent for 5min, and then drop neutral gum to seal the piece.



Staining result:

Senescent cells are blue.


Legend:

图片1

Fig.1 Wi-38 cells were stained by β-galactoside kit. The figure on the left shows the senescent Wi-38 cells without division and proliferation ability but still alive. The positive staining cells after staining are more than 95%.The image on the right shows newly resuscitated WI-38 cells with less than 3 passes (Early Passage) and no significantly positive cells after staining.



Notes

       1. β-galactosidase staining fixation solution is toxic and corrosive, so please pay attention to protection during operation.

2. X-Gal solution should be defrosted completely at room temperature and mixed before use.β-galactosidase staining solution A and B should be restored to room temperature before use.The prepared dyeing working solution should be fully mixed without precipitation before use.

3. The β-galactosidase staining reaction of senescent cells depends on specific pH conditions and cannot be performed in a CO2 incubator

 The β-galactosidase staining reaction of senescent cells depends on specific pH conditions and cannot be performed in a CO2 incubator Incubate to develop color, otherwise it will affect the pH of the dyeing working fluid, resulting in dyeing failure.

4. Choose polypropylene or glass consumables when preparing dyeing solution

5. Color development time 2h- overnight, during which the color development should be observed several times, too short time may lead to negative results;Too long can lead to false positives.The color developing time is closely related to the amount of β-galactosidase contained in the sample itself.

6. β-galactosidase staining of tissue sections requires higher preparation of samples. The samples should be stored at -80℃ and the experiment should be completed in a short time as soon as possible.Because β-galactosidase is very easy to inactivate, improper storage of the sample or too long time can lead to inactivation of the enzyme, staining is not positive.

7. Before preparing the dyeing solution, the pH value of dyeing solution A should be checked. If it is not 6.0(the pH may change due to the preservation conditions), HCl or NaOH should be used to adjust the pH to 6.0 before use.


Packing and Shipping

All of our products will be packed in standard carton.

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