Q: What is the purpose of the Pretreatment Solution?
A: There are two main types of β-galactosidases in senescent cells: SA-β-galactosidase and Lysosomal β-galactosidase. Lysosomal β-galactosidase activity can only be detected at pH 4. The Cell Pretreatment Solution is used to induce lysosomal alkalinisation to minimize the Lysosomal β-galactosidase activity.
Q: Why does the Pretreatment Solution appear to be toxic to my cells?
A: The Pretreatment Solution is non-toxic to cells and should not induce cell death; however some cell types do experience morphology changes after treatment. Despite changes in morphology, the SA-b-galactosidase activity should not be affected.
Q: How long does the fluorescence last?
A: The fluorescence will last for several hours to one day.
Q: Can this assay be used with cells that express GFP?
A: This product is not recommended for use on GFP expressing cells because of the emission spectra overlap between GFP and the fluorogenic substrate. If you are using GFP expressing cells, you may want to consider using either our 96-Well Senescence Activity assay #CBA-231 or our Senescence Staining kit #CBA-230, although these kits are not suitable for analysis by flow cytometry.
Q: How long can the cells be saved until analyzing with flow cytometry?
A: We recommend analyzing the results within a few hours because this assay uses live cells and if the cells lyse the dye will leak out.
Q: What is the best incubation time, 4 hours or overnight?
A: An overnight incubation will give better results, especially with cells that may have lower SA-ß-Gal activity.
Q: Do I need to incubate the cells with 5% CO2?
A: This assay is performed on live cells, so the incubation steps should be performed under their normal growth conditions. If you typically grow your cells with 5% CO2, this should be maintained during this assay. The CO2 environment of the incubator will not affect the pH of the assay.
Q: Can this be used with non-adherent cells?
A: This kit can be used on any mammalian cells, including suspension cells. The only protocol modification required is to spin down the cells before aspirating the media in step 1. The rest of the protocol is the same.