The following assays were performed as previously described.14 (link) Briefly, 50B11 (Sirt2-WT cell line with vector and Sirt2-KO obtained via CRISPR/Cas9 gene editing) cells were seeded in 6-well plates. After induction of differentiation for 24 hours,27 (link) cells were treated with cisplatin at 1 and 2 µg/mL. H1299 and SCC-25 cells were plated and treated with 1 and 2 µg/mL cisplatin. Cell survival was assessed at 24, 48, and 72 hours after cisplatin treatment. For NAD+ treatment, differentiated 50B11, H1299, and SCC-25 cells were pretreated with 5 µM NAD+ for 1 hour followed by cisplatin treatment. At the end of the experiments, 6-well plates were kept on ice, and the number of live cells from each sample was determined using an Automated Cell Counter (Bio-Rad, TC20) after trypan blue staining. The survival fraction was calculated as the number of cells that survived after drug treatment normalized to the number of cells that survived after vehicle treatment times 100.
Automated cell counter
Manufactured by Bio-Rad
Sourced in United States, Singapore, Germany, United Kingdom
The Automated Cell Counter is a laboratory instrument designed for the automated counting and analysis of cells. It provides accurate and reproducible cell counts, enabling efficient and reliable data collection for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue solution, by Merck Group (7 mentions)
Trypan blue, by Merck Group (5 mentions)
Roswell park memorial institute (rpmi), by Corning (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Trypsin edta, by Merck Group (4 mentions)
Histopaque histopaque 1077 cell separation medium, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (4 mentions)
Counter slides, by Bio-Rad (3 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Trypan blue, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Trypan blue, by Bio-Rad (2 mentions)
Bms309403, by Santa Cruz Biotechnology (2 mentions)
Puromycin, by InvivoGen (2 mentions)
Accutase, by Innovative Cell Technologies (2 mentions)
70 μm strainer, by BD (2 mentions)
Ficoll, by Cytiva (2 mentions)
Roswell park memorial institute (rpmi), by Bio-Rad (2 mentions)
134 protocols using automated cell counter
1
Cisplatin Cytotoxicity Assay with NAD+ Pretreatment
2
Curcumin Modulates Fibrocyte Chemotaxis
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Twenty-four-well chemotaxis chambers (Corning, USA) were used in the chemotaxis assays. The chambers were balanced in serum-free medium for 1 hour before seeding the cells. CCL21 (PeproTech, USA) and various concentrations of curcumin were added in the bottom wells of the chamber to function as the chemotactic stimuli. Human circulating fibrocytes (pretreated with different concentrations of curcumin for 72 hours) were collected and suspended in 100 μl of 2% BSA in DMEM, and 1×10 6 viable cells were plated in the top wells of the chamber. Cells were allowed to migrate for 3 hours at 37°C in a 5% CO2 incubator. After migration, the cells on the upper surface of the filter were wiped and the cells sticking to the lower surface were collected and counted using an Automated Cell Counter (Bio-Rad, USA). The chemotaxis
3
Cardiomyocyte Proliferation Modulation
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Effects of pioglitazone and the VEGFR-2-selective inhibitor apatinib (Apexbio
Technology LLC, Houston, TX, USA) on cardiomyocyte proliferation were measured
by counting crystal violet-stained cells 24 h after treatment using an automated
cell counter (BioRad). Briefly, cardiomyocytes (5 × 104cells/well) were seeded in 96-well plates and cultured in standard medium for 24
h. After 24 h serum starvation, cardiomyocytes were treated with 0.1
µM angiotensin (Ang) II for 24 h. Pioglitazone (0,
10 or 20µM) and apatinib (2 µM)
was added to the culture medium 2 h prior to Ang II administration. For crystal
violet staining, the cells were washed twice with 1× phosphate-buffered
saline, fixed with 20% methanol for 30 min, and stained with 0.2% crystal violet
solution for 30 min at room temperature with gentle shaking. Stained cells were
washed with water until a clear background was visible. Crystal violet dye was
extracted using 1% SDS and the cells were counted using an automated cell
counter.
Technology LLC, Houston, TX, USA) on cardiomyocyte proliferation were measured
by counting crystal violet-stained cells 24 h after treatment using an automated
cell counter (BioRad). Briefly, cardiomyocytes (5 × 104cells/well) were seeded in 96-well plates and cultured in standard medium for 24
h. After 24 h serum starvation, cardiomyocytes were treated with 0.1
µM angiotensin (Ang) II for 24 h. Pioglitazone (0,
10 or 20µM) and apatinib (2 µM)
was added to the culture medium 2 h prior to Ang II administration. For crystal
violet staining, the cells were washed twice with 1× phosphate-buffered
saline, fixed with 20% methanol for 30 min, and stained with 0.2% crystal violet
solution for 30 min at room temperature with gentle shaking. Stained cells were
washed with water until a clear background was visible. Crystal violet dye was
extracted using 1% SDS and the cells were counted using an automated cell
counter.
4
SIRT1 Knockdown and Cellular Senescence
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HFK cells with and without stably transfected HPV31 genomes as well as CIN612 cells were stably transduced as described above. After selection, 2x105 passage-matched wild-type, shGFP, and shSIRT1-5 cells were plated in 60mm dishes with J2 feeder cells. When wild-type or shGFP cells reached 80% confluence, all cells were treated with Versene to remove feeders and harvested. Harvested cells were stained with Trypan Blue (BioRad) and counted using an automated cell counter (BioRad) and viability and cell number were determined. Harvested cells were serially passaged in the same manner until approximately day 30 or until natural senescence in the case of primary HFKs. Population doubling of SIRT1 knockdown and control cells was determined as previously described using the following equation:
PD = (logNf–logNi)/log2, where Nf is the number of cells counted after harvesting, and Ni is the number of cells seeded [44 (link)].
PD = (logNf–logNi)/log2, where Nf is the number of cells counted after harvesting, and Ni is the number of cells seeded [44 (link)].
5
Lactate production in EGF-treated cells
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In total, 1 × 106 cells were plated in 6‐well plates and treated with 20 ng/mL EGF, 10 μmol/L SB203580, 10 μmol/L 2‐DG alone or in combinations for 24 h, and the cell culture supernatants and cells were collected separately. Next, the lactate concentration in the culture supernatant was determined using the Lactate detection kit (#A019‐2‐1, Nanjing Jiancheng Bioengineering, Nanjing, Jiangsu, China) according to the manufacturer's protocol. Harvested cells were stained with trypan blue, and viable cells were counted using an automated cell counter (Bio‐Rad, Hercules, CA, USA). Then, the adjusted lactate concentration was calculated according to the following formula: adjusted lactate concentration = lactate concentration in experimental group / the ratio of cell number in the experimental group to that in the control group.
6
Isolation and Expansion of Rat Neural Stem Cells
7
Splenocyte Stimulation with Stroke Serum
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Whole splenocyte culture was performed as previously described.12 (link) Spleens from naïve wild-type mice (C57BL/6J) were dissected and single splenocyte suspension prepared by mincing and using a 40 μm cell strainer. Cells were washed with PBS, cell numbers and viability was assessed using an automated cell counter (Biorad, Germany). The required viability threshold was ≥80%. Cells were then cultured (RPMI 1640, 10% (v/v) heat-inactivated FBS, 1% (v/v) penicillin/streptomycin, 10 μM β-mercaptoethanol) on a 96 well flat bottom plate at a density of 100,000 cells per well in a final volume of 200 μL. Prior to the stimulation, cells were stimulated with serum from either stroke or sham operated mice at a concentration of 25 % (v/v) total well volume for 16 h. After stimulation, cell numbers were analysed by flow cytometry using antibodies against CD45, CD3 and CD11b.
8
Senescent FAPs Secretome Analysis
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Conditioned medium was collected from either non‐senescent or senescent FAPs in DMEM 4.5 g/l glucose supplemented with 10% FBS and 1% penicillin–streptomycin‐glutamine at 37°C at 3% O2 incubator for 24 h. Conditioned medium was filtered through 0.22 μm filter before analysis, and the cell number was quantified using an automated cell counter (Biorad) for normalization. The concentrations of GDF‐15, TNFRI, TNFRII, IL‐6, IL‐10, ICAM‐1, PAI‐1, Eotaxin, MCP‐1, MMP‐2, and OPN in 50 μL of conditioned medium were quantified using multiplex magnetic bead immunoassays (R&D Systems).
9
Lung Lavage and Macrophage Analysis
10
Cell Proliferation and Survival Assays
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