Table 4 ∣.

Troubleshooting table

Step	Problem	Possible reason	Solution
5	Low percentage of EdU+ cells in control group	Cells have low proliferative capacity	Prolong the incubation time with EdU
9	False negative staining	Insufficient X-gal	Ensure you are using the right concentration of X-gal
		Insufficient time of incubation	Titrate the time for X-gal solution incubation
		pH of X-gal solution is >6.0	Add appropriate amounts of citric acid or sodium phosphate buffer to adjust the final pH
	False-positive staining	pH of X-gal solution is <6.0	Add appropriate amounts of citric acid or sodium phosphate buffer to adjust the final pH
		Improper confluence of cells	Make sure cells are 50-70% confluent
30A(xxvii),30B(xxvi),30C(viii),30D(ix), 82A(xxii),82B(xxiii)	Absent or weak signal	Low duration of GL13 reagent incubation	Increase incubation time of GL13 reagent in a step-wise manner
			Incubate GL13 reagent at 37 °C. Increasing the temperature from RT to 37 °C might result in enhancement of the GL13-LF interaction and eventually to a stronger signal
		Insufficient penetration of GL13 reagent into the cells	Use a permeabilization step before the addition of the GL13 reagent. Incubate with 0.5% (vol/vol) Triton X-100/TBS for 5 min at 4 °C. Then continue with next steps
		Reduced anti-biotin antibody reaction	Increase incubation time with anti-biotin antibody
			Increase anti-biotin antibody concentration. (Decrease the dilution of the anti-biotin solution)
	High nonspecific background staining	Insufficient washing	Thoroughly wipe away excess GL13 reagent immediately after the incubation step is complete
			Increase washing time in ethanol and TBS solutions. Add several wash steps either to remove excess of GL13 (ethanol solution) or to avoid non specific reaction of the anti-biotin antibody and the detection system (TBS solution)
		Duration of GL13 reagent incubation is too long	Reduce GL13 reagent incubation time in a stepwise manner
		High concentration of the anti-biotin antibody	Decrease the anti-biotin antibody concentration. (Increase the dilution of the anti-biotin solution)
			Decrease the anti-biotin antibody incubation time
		High concentration of the secondary antibody	Decrease the concentration of the secondary antibody. (Increase the dilution of the secondary antibody)
		Increased DAB exposure	Reduce DAB exposure time
35,43	No amplification detected	Poor RNA quality or improper qPCR settings	Make sure that the RNA is not degraded and the protocol for cDNA amplication is correct
51	BCA shows high background and poor standard curve	Phenol red still present in the cell culture medium	Repeat collection of conditioned medium in phenol red-free medium, or thoroughly buffer exchange the sample
52	Albumin is one of the major proteins detected by mass spectrometry	Serum proteins (from FBS) are still present in the cell culture medium	More thoroughly wash cells with PBS prior to moving cells into serum-free medium (for collection of secreted proteins)
	Many proteins are more abundant in control samples	Non-senescent (control) cell counts are significantly greater than senescent cell counts	Normalize the sample loading or protein quantification to cell counts
53	False-negative staining	The enzyme activity of SA-β-Gal is lost	Make sure that the tissues are fresh and stain them immediately done after sectioning
58	False-positive staining	Overstaining	Make sure that the staining time is not too long, otherwise false positive signals can be detected in the control
85	False-negative staining	Antigen retrieval is not done properly	Optimize the antigen retrieval