Senescence β galactosidase staining kit 9860

To measure SA-β-gal activity, 6-well Nunc plates, which had been coated with 1% gelatin and washed with 1x PBS, were seeded with 1 × 10⁵ cells per well. After removing the medium, the cells were stained using an x-Gal staining kit (Senescence β-Galactosidase Staining Kit # 9860; Cell Signaling Technology, http://www.cellsignal.com) to determine the number of senescent cells, following the manufacturer's protocol. After staining, images from five random microscopic fields were acquired by using a light microscope (OLYMPUS, Tokyo, Japan).

Beta nicotinamide mononucleotide (β-NMN) (BTO5) was purchased from BONTAC (Shenzhen, China). Nicotinamide phosphoribosyltransferase (Nampt) inhibitor (GMX1778) was from Selleck (Shanghai, China). Lomotil (Atropine-diphenoxylate) was purchased from Kangpu Pharmaceutical Ltd Co (Changzhou, China). Antibodies against serotonin transporter (ab181034), tryptophan hydroxylase (ab52954), Lgr5 (ab75850) and Nampt (ab45890) were purchased from Abcam (Cambridge, MA, USA). SR4 (sc-376158), p15/16 (sc-377412) and goat anti-mouse IgG-FITC (sc-2010) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). APC anti-mouse CD117 (c-kit) (135108) was purchased from BioLegend (San Diego, CA, USA). Proliferating cell nuclear antigen (2586), GAPDH (5174) and Senescence β-Galactosidase Staining Kit (9860) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse IgG (31430), goat anti-rabbit IgG (31460) and TRIzol Reagent (15596-018) were purchased from Invitrogen (Carlsbad, CA, USA). EvaGreen Supermix (172-5201AP) was purchased from Bio-Rad Laboratories Inc (Hercules, CA, USA). PrimeScript first strand cDNA Synthesis Kit (6110A) was purchased from TaKaRa Bio Inc (Dalian, China). Nuclear Fast Red solution (N3020) and all other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise stated.

U87-MG cells were seeded into 24 well plates at 5 × 10⁴ cells per well, and then treated with FL3 for 48 h. Afterwards, the treatment was stopped, cells were washed with PBS, refilled with new fresh medium and incubated for 8 days. Medium was changed every two days. At each time point (2, 4, 6 and 8 days), an aliquot of the cells was fixed and stained with the senescence β-galactosidase staining kit #9860 (Cell Signaling), and then kept in a dry incubator (without CO₂) at 37 °C overnight. Cells were checked under an inverted microscope (Nikon Eclipse TS100, Nikon) for blue color detection and photos were taken using the NIS element software (Nikon). We did the same protocol of SA-β Gal staining on U87-MG (TMZ off), U373-MG and LN443 cells.

SA β-gal staining of kidneys, livers, and brains was performed per kit instructions (Cell Signaling Senescence β-Galactosidase Staining Kit #9860). Briefly, tissue samples were snap-frozen in an optimal cutting temperature compound Tissue-Tek® OCT™ Compound (Sakura Finetek Europe B.V., The Netherlands). Fresh frozen sections (10-µm thick) were cut with a cryostat (Leica Biosystems, Germany), and mounted on slides. The slides were fixed for 15 minutes in 1x fixative solution, followed by PBS washes, and incubated overnight with beta-galactosidase staining solution in a dry incubator at 37 °C with no CO₂ supply to prevent false positivity. Sections were viewed under a microscope for blue color development and were photographed with an Olympus BX41 camera^{59 (link)}. After photographing the slides, positive cells for SA β-gal were counted using ImageJ software (ver. 1.38; National Institutes of Health, USA).

Cells were seeded in six-well plates at a density of approximately 20 000 cells/well before treatment with paclitaxel for 48 h. After culture for a further 5 days, cells were fixed and stained using a Senescence β-Galactosidase Staining Kit #9860 purchased from Cell Signalling Technology (Beverley, MA, USA). Plates were incubated overnight at 37 °C in a dry incubator (no CO₂). Cells were then detected for blue staining under a bright-field microscope. The percentage of SAβ-gal-positive cells was calculated by counting the cells in five random fields.