1. Experimental issues and precautions

Q1: No obvious blue reaction was observed in SA-β-gal staining

- Possible reasons: pH of the staining solution deviates from 6.0 or incubation time is insufficient.

- Suggestion: Calibrate the pH of the staining solution to 6.0, extend the incubation time and observe.

Question 2: Principles of negative control setting

- Select young or non-senescent healthy cells, treat them under the same conditions as the experimental group, and verify background staining.

2. Construction of cell senescence model and β-Gal staining identification method

Method 1: Replicative aging model (taking bladder smooth muscle cells as an example)

• Materials and culture:

- Cells: bladder smooth muscle cells; culture medium: DMEM/F12.

- Subculture operation: When the cell fusion reaches 80%-90%, trypsin digestion and subculture at a ratio of 1:2. The population doubling times are calculated by the formula PD=3.32×log(NH/N0). Senescent cells are obtained when subcultured to PD 30-50 (N0 is the number of cells plated, and NH is the number of cells collected).

• SA-β-gal staining steps:

1) Discard the culture medium and wash 3 times with PBS;

2) Fix with fixative at room temperature for 15 min;

3) Wash 3 times with PBS;

4) Add the staining solution preheated at 37℃ and incubate in the dark for 3-16 h (seal with plastic wrap to prevent evaporation);

5) Count the blue positive cells under an optical microscope and store at 4℃ (PBS or mounting medium).

Method 2: D-galactose-induced aging model

3. Core biological characteristics of cellular aging

• Cell cycle arrest: permanent exit from the cell cycle, manifested as G1/S or G2/M phase arrest.
• Morphological changes: cell volume increases, cytoplasmic vacuolization, and significant differences from normal cells.
• Telomere shortening: Telomere length detection is one of the classic markers of aging cells.
• Senescence-associated heterochromatin colonies (SAHF): chromatin condenses to form dispersed and dense heterochromatin particles.
• Senescence-associated secretory phenotype (SASP): secretes inflammatory factors, matrix metalloproteinases, etc., to reshape the microenvironment.
• Changes in molecular markers: The activity of senescence-associated β-galactosidase (SA-β-gal) increases, catalyzing the substrate to produce a dark blue product, which is the most commonly used aging detection indicator.

The answer to this question comes from MCE Technical Support.

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