Cell Aging β - Galactosidase Staining Kit

Cat. No.: C1342264

GRADE & PURITY BioReagent, for Microscopy

Overview

Cell senescence refers to a progressive degenerative process in cells. Over time, their capacity for proliferation and differentiation, as well as physiological functions, gradually decline during vital activities. Normally, senescent cells are cleared by the organism. Unremoved senescent cells may accumulate in the body, triggering low-grade inflammation, and further inducing deterioration of adjacent tissues or even carcinogenesis. Typical characteristics of cell senescence include altered cell morphology, cell cycle arrest, senescence-associated secretory phenotype, macromolecular damage and metabolic disorders. At present, senescence-associated β-galactosidase (SA-β-Gal) is the most specific biomarker for identifying cell senescence. Its level gradually rises as cells senesce, while it is undetectable in pre-senescent cells, quiescent cells and terminally differentiated cells.

This kit takes advantage of the property that β-galactosidase in senescent cells can catalyze the hydrolysis of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) at pH 6.0 to produce blue precipitates. The senescent status of cells or tissues can therefore be observed under a light microscope.

 Product Components

C1342264	Component	100T	Storage
C1342264A	β-Galactosidase Staining Fixative	100 mL	-20℃. .Avoid freeze/thaw cycle.
C1342264B	β-Galactosidase Staining Solution A	1.5 mL	20℃. Avoid freeze/thaw cycle.
C1342264C	β-Galactosidase Staining Solution B	1.5 mL	-20℃. Avoid freeze/thaw cycle.
C1342264D	β-Galactosidase Staining Solution C	100 mL	-20℃. Avoid freeze/thaw cycle.
C1342264E	X-Gal Solution	5 mL	-20℃. Avoid freeze/thaw cycle. Store in the dark.

Storage Conditions

Store all components at -20 °C. Component E (X-Gal Solution) requires protection from light.

Taking adherent cells cultured in 6-well plates as an example, add 1 mL of staining working solution per well. A total volume of 100 mL is sufficient for 100 wells.

Instructions for Use

1. Staining Protocol for Adherent Cells (6-well Plate)

①Seed cells and incubate overnight. Perform the experiment when cell confluence reaches 60%–80%.

②Discard the culture medium and rinse cells once with PBS.

③Add 1 mL of β-galactosidase staining fixative, and incubate at room temperature for 10–15 minutes for fixation.

④Remove the fixative, then rinse with PBS three times, 2–3 minutes each time.

⑤Prepare the staining working solution according to Table 1. Calculate the total volume based on sample type and quantity.

Table 1. Preparation of Staining Working Solution

Components	Preparation for 1 mL working solution
β-Galactosidase Staining Solution A	10 µL
β-Galactosidase Staining Solution B	10 µL
β-Galactosidase Staining Solution C	940 µL
X-Gal Solution	40 µL

Notes: To reduce crystal formation during staining, warm X-Gal solution at 37 °C for 1–3 hours before use. Prepare the staining working solution freshly before use and consume it within 15 minutes.

⑥Aspirate PBS, add 1 mL of staining working solution to each well, and incubate overnight at 37 °C in a CO₂-free incubator.

Note: To avoid staining artifacts caused by liquid evaporation, fill the peripheral wells with water or PBS to minimize the edge effect. Seal the plate with parafilm or plastic wrap.

⑦Observe and count cells under a conventional light microscope.

⑧Optional step: Remove the staining working solution, add 2 mL of PBS. Samples can be stored at 4 °C for several days.

2. Staining of Suspension Cells

①Harvest cells and centrifuge at 2500 ×g for 10 minutes, then rinse once with PBS.

②Add 1 mL of β-galactosidase staining fixative and fix cells at room temperature for 10–15 minutes.

Note: Gently shake the cells on a shaker to prevent cell clumping.

③Centrifuge at 2500 ×g for 10 minutes and discard the fixative. Rinse cells with PBS three times, 1–3 minutes each time.

④Prepare the staining working solution according to Table 1. Calculate the required total volume based on sample type and quantity.

⑤Centrifuge at 2500 ×g for 10 minutes and remove PBS. Add 0.5–1 mL of staining working solution to each tube and incubate overnight at 37 °C.

⑥Pipette an aliquot of cell suspension onto a glass slide or into a 6-well plate, then observe and count cells under a standard light microscope.

⑦Optional: Remove the staining working solution and add 1 mL of PBS. Samples can be stored at 4 °C for several days.

Precautions

①Briefly centrifuge all reagents to spin down liquid to the bottom of tubes before use.

②Senescence-associated β-galactosidase staining requires a specific pH environment. High CO₂ concentration in a CO₂ incubator will alter the pH of the staining working solution. Therefore, incubation at 37 °C must be performed in a CO₂-free incubator.

③X-Gal solution will freeze at -20 °C or 4 °C. Thaw completely by water bathing at room temperature or 37 °C for 2–5 minutes with gentle shaking. Use polypropylene (PP) or glassware for aliquoting X-Gal solution or preparing working solution. Do not use polystyrene (PS) consumables such as cell culture plates and serological pipettes.

④A small amount of precipitate may form at the bottom of the tube containing β-galactosidase Staining Solution B, which is normal. Vortex thoroughly until fully dissolved prior to staining.

⑤For tissue section staining, refer to relevant literature and optimize the protocol through preliminary experiments.

⑥The β-galactosidase staining fixative is toxic and corrosive. Wear a lab coat, disposable mask and gloves during operation. Avoid direct skin contact and inhalation.

Storage and Shipping

Storage

Protected from light,Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (12 months). Avoid freeze/thaw cycle. Store in the dark.