Senescence cell histochemical staining kit

Cells were stimulated with TGF-β1 10 ng/ml during 72h. The senescence cell histochemical staining kit (Sigma Aldrich; catalogue no. CS0030) based on a histochemical staining for β-galactosidase activity was used. After TGF-β1 stimulation, cells were fixed with the fixation buffer provided by the kit (solution containing 20% formaldehyde, 2% glutaraldehyde, 70.4 mM Na2HPO4, 14.7 mM KH2PO4, 1.37 M NaCl, and 26.8 mM KCl) for 6–7 minutes at room temperature. Cells were stained with the staining mixture provided by the kit and incubated at 37°C without CO₂ overnight. Finally, cells were observed under a microscope to count the blue-stained cells and the total number of cells x field. Results were expressed as % senescence (β-galactosidase blue positive cells) relative to the total number of cells in each field.

After 120 hours of co-culture, cells were washed with PBS and fixed in fix solution before the staining with X-gal solution according to the manufacturer’s instructions (Senescence Cell Histochemical Staining Kit, Sigma-Aldrich). After 24 hours of incubation at 37°C. DAPI was applied to stain the nucleus [14 (link)].

Cells were observed with a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Europe B.V., Amsterdam, Netherlands) coupled to a XM Full HD digital camera (Hangzhou Xiongmai Technologies (XM), Hangzhou, China). iMSC proliferation was calculated as cumulative population doublings (PDs) at each passage, following the formula in Equation (1), where Nf is the final cell number, Ni is the initial cell number, and log is the natural logarithm [46 (link)]. The number of accumulated generations per days in culture was analysed by regression for each cell line. Generation time was calculated for each cell line at each passage as the number of PDs per day, and generation times of all cell lines were compared.
$\begin{array}{c}\text{PD}=\frac{\text{logNf}‐\text{logNi}}{\log 2},\end{array}$
Histochemical staining for senescence-associated β-galactosidase activity was performed for each cell line after reaching more than 100 PDs at three different passages, using the Senescence Cell Histochemical Staining kit (Sigma-Aldrich Química S.A.). After 16 hours of incubation, β-galactosidase-positive and β-galactosidase-negative cells were counted on ten random microscope fields and percentage of senescent cells was calculated. Results are provided as mean percentage of senescent cells ± standard error. Senescence values of iMSCs and primary MSCs were compared.

Cell morphology of hASCs-M, hASCs-T, hASCs-TS and hASCs-TE was observed and analyzed using a phase contrast microscope (CKX41, Olympus, Tokyo, Japan). Cells were than photographed at 10X magnification with a C-7070 digital compact Camera (Olympus,).
Senescence-associated β-galactosidase activity was evaluated with a senescence cell histochemical staining kit (SIGMA Aldrich, USA), according to protocol instructions. Briefly, 3.0 × 10³ cells/cm² were plated in 12-well tissue culture plates (in triplicate) and incubated at 37°C, 5% CO₂ for 48 hours. Cells were then washed twice with 1 ml/well PBS 1X and fixed for seven minutes at room temperature. After three washes, cells were stained at 37°C overnight and visualized under a microscope.

The MSCs were stained for β-galactosidase, a hydrolase enzyme that specifically catalyses the hydrolysis of β-galactosidase into monosaccharides in senescent cells. MSCs were analysed for the expression of β-galactosidase using the Senescence Cell Histochemical Staining Kit (Sigma-Aldrich). MSCs at passage 8 were seeded at 4000 cells/cm² in duplicates in 12-well plates (Corning, New York, USA) and kept in culture until 50–70% confluence when the cells were fixed and stained according to the manufacturer’s instructions. The number of cells that stained blue (positive for β-galactosidase), and the total number of cells were counted in five random view fields per well at 10× magnification, and the percentage of β-galactosidase positive cells was calculated.

SKOv3-ARHI and Hey-ARHI cells were grown in 6-well plates at an initial density of 20 000 cells per well. After 48 h of incubation with DOX to induce ARHI expression, cells were washed with PBS and fixed in 4% paraformaldehyde before staining with X-gal solution according to the manufacturer's instructions (Senescence Cell Histochemical Staining kit, Sigma-Aldrich). After cells were incubated in the staining solution for 16 h at 37 °C, β-galactosidase-positive cells with blue precipitate were counted using bright-field microscopy.

The degree of senescence was determined using the mammalian β-Gal assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Following 2 weeks of cultivation, cells were washed twice with cold PBS and collected for protein extraction using M-PER mammalian extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA). An equivalent amount of β-Gal assay reagent was added to 50 μL of the supernatant obtained after centrifugation at 15,000 rpm for 15 min. Following incubation for 30 min at 37 °C, absorbance was measured at 405 nm using a Synergy H2 microplate reader (BioTek, Winoosk, VT, USA). The experiment was performed in triplicate, and the data are presented as the percentage of absorbance values.
Microscopy images of cells were stained using the senescence cell histochemical staining kit (#CS0030; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Following 1 week of cultivation, cells were washed twice with cold PBS, fixed with 1 mL 4% formaldehyde solution (Wako, Kyoto, Japan) and incubated for 2 h at room temperature. To visualize the physiological changes, images and number of β SA-β-Gal stained cells in a given area were obtained using an optical microscope, IX-75 (Olympus, Tokyo, Japan).