The cell proliferation and viability to exogenous stimuli were spectrometrically quantified by using WST-1 reagent (Roche, Basel, Switzerland) according to the manufacturer’s instructions. A total cell number of 1700 per cavity of a 96-well culture plate (Greiner bio-one, Frickenhausen, Germany) were used and maintained in fully supplemented growth medium for 24 h before treatment with LPS and ATP. After 20 h, the WST-1 reagent was added to each well. The absorbance at the wavelength 440 nm and 590 nm as a reference were determined at time points 0, 1, 2, 3, and 4 h after WST-1 supplementation using a multiplate reader (Tecan, Männedorf, Switzerland).
96 well culture plate
Manufactured by Greiner
Sourced in Germany, United Kingdom
The 96-well culture plates are a standard laboratory tool designed for cell and tissue culture applications. They provide a grid of 96 individual wells, allowing for the simultaneous culturing and analysis of multiple samples or experimental conditions.
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Lab products found in correlation
Rpmi 1640 medium, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Infinite f200 pro, by Tecan (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Wst 1 reagent, by Roche (1 mentions)
Multiplate reader, by Tecan (1 mentions)
Accutase, by PAN Biotech (1 mentions)
Bioscience influx sorter, by BD (1 mentions)
Resazurin, by Merck Group (1 mentions)
Synergy h4 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Celltiter glo 3d cell viability assay, by Promega (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Recombinant human il 2, by BD (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Human serum, by Merck Group (1 mentions)
24 well culture plate, by Corning (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Penicillin, by Merck Group (1 mentions)
19 protocols using 96 well culture plate
1
Cell Proliferation Quantification by WST-1
2
Single-cell Cloning of Adipogenic and Osteogenic Cells
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Adipogenic and osteogenic differentiation studies were conducted as previously described.9 In addition, single‐cell cloning was performed on 2 sAPC lines at P3, using a motorized device connected to the flow cytometric sorter (Cyclone, Beckman Coulter). Sorted cells were placed into each well of a 96‐well culture plate (Greiner Bio‐one, UK) and cultured up to 4 weeks in endothelial cell growth medium‐2 for quantification of colonies generated from a single cell.
3
Monocyte Isolation and Stimulation
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Monocytes were harvested directly after isolation and after 24 h stimulation with GM-CSF and adjusted to a final concentration of 1 × 106 cells/mL. Subsequently, 100 µL of cell suspension were seeded per well (in triplicate) and incubated for 2.5 h at 37 °C and 5% CO2 under static conditions using a 96-well culture plate (Greiner-Bio-One, Leipzig, Germany). Supernatants were decanted, and adherent cells were washed with 100 µL PBS per well. Adherent cells were detached by incubation with accutase (100 µL per well) (PAN-Biotech, Aidenbach, Germany) at room temperature for 20 min. Triplicate samples were pooled in a 1.5 mL tube, and the reaction was stopped by adding the cell culture medium to a final volume of 1 mL. Monocytes were then counted using the CASY cell counter.
4
Clonogenic Potential of Progenitor Cells
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To test the ability of CPs to generate colonies starting from a single cell, CPs (n=4) at P5 were selected as Propidium Iodide negative (live cells) and sorted into single cells using a BD Bioscience Influx sorter (BD Biosciences, UK). Sorted cells were placed into each well of a 96-well culture plate (Greiner Bio-one, UK). The sorted cells were cultured up to 4 weeks in EGM-2 and the number of colonies generated was counted.
5
Cell Viability Assay on Hydrogels
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Cells were seeded in a 96 well culture plate (Greiner) at a density of 2 × 103 cells/well on hydrogels or polystyrene in full DMEM/F12 media for 5 days at 37 °C, 5% CO2. After treatment, viability was measured by trypan blue staining. The metabolic activity analysis was performed using MTT tetrazolium dye. MTT was added to each well at a final concentration of 0.5 mg/mL, and the cells were further incubated at 37 °C for 4 h. Lysis buffer (isopropanol solution with 1% NP-40, 4 mM HCl) 150 μL was added to each well after removing the medium. After resuspension and 30 min incubation, the plate was centrifuged at 1000× g for 10 min. The supernatant was transferred to new wells, and the absorbance was measured at 595 nm using a microplate absorbance reader (iMark, Bio-Rad, Hercules, CA, USA).
6
SH-SY5Y Neuronal Cell Viability Assay
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SH-SY5Y cells were seeded on a 96-well culture plate (Greiner Bio-One) at 1.0 × 10⁵ cells/ml using Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were cultured overnight and adhered to the plate. The medium was replaced with a culture medium containing sesaminol and/or 6-OHDA at various concentrations, and the cells were cultured in an incubator for various times. When sesaminol and 6-OHDA were used together, sesaminol was added 2 h before the addition of 6-OHDA. After the treatment, the medium was removed, and 100 μl of the culture medium containing 10% of a 5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well. The cells were left in the incubator for 2 h. The medium was removed, and 200 μl of DMSO was added to each well. The culture plate was stirred for 3 min on a plate mixer (Biotec, Tokyo, Japan), and the absorbance at a wavelength of 535 nm was measured using a microplate reader (Wallac 1420 ARVO sx).
7
Cell Adhesion and Proliferation Assay
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Two hundred microliters of cell suspension at a density of 10 3 cells/well was seeded onto each specimen in a 96-well culture plate (Greiner Bio-One North America Inc.). The cells were allowed to adhere to the surface of the specimens for 45 min (10 μL of cell suspension) before adding the rest of the culture medium (190 μL) [3] . A pilot study conducted before testing had shown that 45 min was a suitable time to allow cell adhesion and prevent cell suspension dehydration. All culture plates were then incubated in 5% CO 2 at 37 ± 1°C, 95% humidity for either 24 h or 72 h.
8
Cell Viability Assays for 3D Cultures
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Cell viability assay was performed on cells cultured in 96-well plates, incubated for 3 h in the dark, at 37 °C in a humidified atmosphere with 5% CO 2 , with a buffered solution that contains highly purified resazurin (Sigma-Aldrich). Cell viability was measured as turnover of resazurin to fluorescent resorufin by metabolically active cells. Fluorescence (excitation 530 nm/emission 590 nm) was detected with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments Inc., VT).
Alternatively, cell viability was determined by quantification of ATP present in metabolically active cells, using the CellTiter-Glo 3D cell viability assay (Promega, WI). The assay was performed according to the manufacturer's instructions in 96-well culture plates (Greiner Bio-One). For quantitative analysis, an ATP standard curve was generated immediately prior to adding the CellTiter-Glo reagent. Luminescence signal was detected using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments).
Alternatively, cell viability was determined by quantification of ATP present in metabolically active cells, using the CellTiter-Glo 3D cell viability assay (Promega, WI). The assay was performed according to the manufacturer's instructions in 96-well culture plates (Greiner Bio-One). For quantitative analysis, an ATP standard curve was generated immediately prior to adding the CellTiter-Glo reagent. Luminescence signal was detected using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments).
9
Cytokine Profiling of M. perstans Infection
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100μl whole blood from M. perstans-infected individuals and NEN were plated onto 96-well culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and cultivated in 100μl RPMI-1640 medium (Sigma-Aldrich, Munich, Germany) including 10% bovine calf serum (BCS, Sigma-Aldrich). Whole blood cultures were then left un-stimulated or re-stimulated with 50μg/ml M. perstans worm extract for 72h at 37°C and 5% CO2. Thereafter culture supernatants were removed and frozen at -20°C until cytokine levels were determined by ELISA.
10
PBMC Isolation and Functional Assays
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Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density centrifugation through Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Cells were cultured at a concentration of 106/ml in RPMI 1640 medium supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine and 10% heat inactivated human AB serum (Sigma-Aldrich, St Louis, MO). Cells were plated in 96 well culture plates (Greiner Bio-One, Germany) and incubated overnight in a 5% CO2 humidified atmosphere at 37°C for rest. Cells were then kept with media alone (unstimulated) or stimulated with either 1 μM of individual peptides or with peptide pools at 1μM/peptide. Recombinant human IL-2 (BD Bioscience, San Diego, CA) at 0.1 ng/ml final concentration was added after 24 hours and every 3 days along the culture period. Cell cultures were incubated for 10 days. As positive control cultures, cells were stimulated with Phytohemagglutinin (PHA) (Sigma-Aldrich, St Louis, MO) (10 μg/ml) and SLA (as indicator of previous disease) (10 μg/ml), for 5 days. Culture supernatants (in triplicate for each condition) were harvested and frozen at -80°C until use for ELISA and CBA assays, whereas cells were used for ELISPOT test and cell phenotyping.
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