Senescence-associated β-gal activity was measured using a Cellular Senescence Plate Assay Kit (Dojindo, Tokyo, Japan) according to the technical manual provided by the manufacturer. Twenty thousand cells were seeded on a black Fluotrac plate (Greiner Bio, Frickenhausen, Germany) and cultured overnight. DNA was stained with Hoechst 33342 and quantified using a Cell Count Normalization Kit (Dojindo, Japan) before the β-gal assay. Fluorescence was measured using SpectraMax M5e (Molecular Devices, Tokyo, Japan). The β-gal activity was normalized to the Hoechst intensity.
Cell count normalization kit
Manufactured by Dojindo Laboratories
Sourced in Japan
The Cell Count Normalization Kit is a laboratory product designed to standardize cell counts across samples. It provides a reliable method to normalize cell concentrations, enabling consistent analysis and comparison of cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m5e, by Molecular Devices (1 mentions)
Rhodamine 123, by Thermo Fisher Scientific (1 mentions)
Rhodamine 123, by Fujifilm (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cellsens standard imaging software, by Olympus (1 mentions)
Cellular senescence plate assay kit spider βgal, by Dojindo Laboratories (1 mentions)
H 1200, by Olympus (1 mentions)
X gal solution, by Merck Group (1 mentions)
Spectramax i3x multi mode microplate reader, by Molecular Devices (1 mentions)
Glucose uptake assay kit, by Dojindo Laboratories (1 mentions)
5 protocols using cell count normalization kit
1
Senescence-Associated β-Gal Activity Assay
2
Evaluating Mitochondrial Biogenesis in Adipogenesis
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Mitochondrial biogenesis was evaluated by rhodamine 123 assay. Cells were seeded in black clear-bottom 96-well plate and induced adipogenesis. At day 14 (D14), the supernatant was replaced by 50 μM rhodamine 123 (Wako, Japan) dissolved in Hanks balanced salt solution (Gibco) containing 20 mM HEPES (100 μL/well) and incubated at 37°C for 30 min. The supernatant was discarded and rinsed with the buffer two times before measuring rhodamine 123 fluorescence intensity [relative fluorescence unit (RFU)] using the excitation/emission at 485/525 nm by microplate reader (Varioskan LUX). RFU was normalized to cell number using Cell Count Normalization kit (Dojindo, Japan). After staining with rhodamine 123, cell nuclei were counterstained with 0.5 μg/mL Hoechst 33342 (Molecular Probes, United States) dissolved in PBS (100 μL/well) for 10 min at room temperature for fluorescent imaging using automated inverted microscope (IX83, Olympus).
3
Quantifying Cellular ATP Production
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ATP production was determined by the “cell” ATP assay luminescent reagent version 2 (CA2-50, Toyo Benet, Japan). Cells were seeded in white clear-bottom 96-well plate, and induced adipogenesis. After 14 days, according to the manufacturer’s protocol, the luminescent reagent (100 μL/well) was added and followed by 1 min on shaker and 10 min for incubation in dark at room temperature. Luminescence amount (RLU) was measured by microplate reader (Varioskan LUX) and normalized to cell number using Cell Count Normalization kit (Dojindo, Japan).
4
Senescence-Associated β-Galactosidase Staining Assay
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Transfer the micro cover glass with the fixed cells from step 4.a.iii. into 24-well plate filled with PBS.
Remove the PBS.
Add 1 mL of Staining mixture including X-gal Solution (Sigma, CS00030).
Seal the plate with Parafilm.
Incubate the cells at 37°C for 16–20 h without CO2.
Wash the cells three times with 1 mL of PBS.
Cover with Mounting Medium with DAPI (VECTASHIELD, H-1200) and observe the cells on DIC and DAPI images with Olympus cellSens Standard Imaging Software using microscope (100× magnification, BX53, Olympus). Count more than 200 DAPI-positive cells manually, and calculate the percentage of SA-β-gal-positive cells per condition in each experiment. Please refer to (Yamamoto-Imoto et al., 2022 (link)).
5
Glucose Uptake Assay of ACSL1-Overexpressing Cells
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ICP2 cells were seeded into 6-well plates and transfected with the pcDNA3.1-EGFP and pcDNA3.1-ACSL1-EGFP plasmids. At 48 h post-transfection, the glucose uptake capacity of the cells was monitored using the Glucose Uptake Assay Kit (Dojindo, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, the cells were washed twice and incubated with pre-heated glucose-free medium (Solarbio) for 15 min at 37 °C. Thereafter, the cells were incubated with the pre-heated probe solution for 15 min at 37 ºC and washed twice with pre-cooled WI Solution (1×). Pre-cooled WI Solution (1×) was added again and incubated at 25 °C for 5 min. Cells were microscopically observed and photographed using an Echo Revolve fluorescence microscope (Echo Laboratories). The total number of cells was determined using the Cell Count Normalization Kit (Dojindo), according to the manufacturer’s instructions. The fluorescence intensity of the cell nucleus (excitation filter: 350 nm; emission filter: 461 nm) and glucose uptake (excitation filter: 545 nm; emission filter: 605 nm) were measured using a SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices Corporation).
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