We used a previously reported MTT assay procedure for lymphocyte proliferation assay (Babu et al., 2008[1 (link)]). Briefly, splenocytes were cultured in 96-well cell culture plates (Nunc, Denmark) and the aliquots of the antigens were then dispensed into the wells, at a final concentration of 10 μg/ml of antigen. Wells with splenocytes from mock-immunized mice were included as negative control. As well as ConA-stimulated cell suspensions were also included as positive control (ConA: Sigma-Aldrich, St. Louis, MO, USA). Cultures were incubated at 37 ºC in 5 % CO2 humidified incubator for 1-3 days. The MTT proliferation assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. After incubation, cells were treated with MTT solvent for 15 minutes at RT. Absorbance was measured at OD = 570 nm.
96 well cell culture plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States, Germany
The 96-well cell culture plates are a lab equipment product designed for standard cell culture applications. These plates provide a standardized and consistent platform for culturing cells in a 96-well format. The plates are made of high-quality, tissue culture-treated polystyrene and are available in various surface treatments to accommodate different cell types and experimental requirements.
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Lab products found in correlation
Minimum essential medium (mem), by Merck Group (3 mentions)
Glomax discover microplate reader, by Promega (2 mentions)
Mc3t3 e1 cell, by American Type Culture Collection (2 mentions)
Celltiter 96 aqueous kit, by Promega (2 mentions)
Resazurin, by Merck Group (2 mentions)
Infinite m2000 pro plate reader, by Tecan (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Phase contrast microscopy, by Olympus (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Brdu cell proliferation assay kit, by Merck Group (1 mentions)
Autolab pgatat302n, by Metrohm (1 mentions)
Live dead baclight viability kit, by Thermo Fisher Scientific (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Fv1000 ix81, by Olympus (1 mentions)
S 4800 sem, by Hitachi (1 mentions)
Tgf β1, by BioLegend (1 mentions)
35 protocols using 96 well cell culture plate
1
Lymphocyte Proliferation Assay using MTT
2
Gentiana Extract Enhances Hyaluronan Production
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Example 3
Gentiana extract (Gentiana acaulis/Gentiana septemfida, 25/75 m/m) was used. Normal human fibroblasts (NHF) P2+1 were seeded at 5000 cell/well in 96 well cell culture plates (Nunclon) in MEM 10% FCS (Pan Biotech). After three days of growth, Gentiana extract solution was added in medium without FCS and incubated the cells for an additional three days. Secreted hyaluronan in the growth medium was measured using a Hyaluronan Assay Kit (K-1200, Echelon, Salt Lake City, Utah).
Compared to untreated cells we showed a 26±4.9% increase in hyaluronan production with 0.001% Gentiana extract and TGFbeta1 (10 ng/ml), served as positive control, stimulated by 45.6±1.1%.
3
Cytotoxicity of Phytoestrogens on Breast Cancer
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Cytotoxic effects of TML and isoflavone reference substances (i.e., formononetin and ononin) were examined on MCF-7 and MDA-MB-231 human breast cancer cells and WS1 cells using an MTT assay. Stock solutions of the tested compounds were prepared via dissolving in a sterile DMSO. The suspension of cells was prepared at a density of 1 × 105 cells/mL and then transferred to 96-well cell culture plates (NUNC, Roskilde, Denmark). After 24 h of incubation, the medium was removed from each well and then cells were incubated for the next 24 h/48 h with different concentrations of the compounds examined. The cytotoxic effect of TML and isoflavones was assessed using the MTT assay. After the respective incubation period (i.e., 24 h or 48 h), MTT solution (5 mg/mL) was added to all wells, and the plates were incubated for 3 h. In the next step, 100 µL of 10% SDS buffer solution per well was added and after an overnight incubation the absorbance was measured at 570 nm using a microplate reader (Epoch, BioTek Instruments Inc., Winooski, VT, USA).
4
Toxicity of SeNPs on H157 Lung Cancer
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To test for the toxicity of SeNPs on cancer cells, a common and fatal cancer, non–small lung cancer cell line H157 was chosen. Cell line H157 bought from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences School of Basic Medicine Peking Union Medical College. H157 cells were grown in RPMI medium with 10% FBS and 1% PEST at 37 °C in a 5% CO2 incubator55 (link). Viability was measured by means of the MTT viability kit in 96-well cell culture plates (Nunc). Cells were counted in a Burke’s chamber, and 10,000 cells per well (200,000 cells/ml) were plated and pre-incubated for 24 h to allow adhesion. After pre-incubation, the medium was discarded, and treated with a series of dosages of SeNPs (0.003, 0.006, 0.012, 0.075, 0.15, and 0.3 μg μl−1 wet weight, in 50 μl RPMI 1640) for 24 h in five replicates. Determination of cells viability was performed according to the operating instructions.
5
Inhibition of Cell Proliferation by 8-Hydroxyquinaldic Acid
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The suspension of HT-29 and LS-180 cells was prepared at a density of 5 × 104 cells/mL and then transferred to 96-well cell culture plates (NUNC). The next day, the culture medium was removed, and the cells were exposed to fresh medium containing 10% FBS (control) or dilutions of 8-hydroxyquinaldic acid (0.0001; 0.001; 0.05; 0.1; 0.5; 1 mM). Proliferation and DNA synthesis was assessed after 48 h by measurement of 5′-bromo-2′-deoxy-uridine (BrdU) incorporation during DNA synthesis, according to the manufacturer’s protocol (Cell Proliferation ELISA BrdU, Roche Diagnostics GmbH, Penzberg, Germany).
6
Toxin Neutralization Potential of Anti-HAB Antibodies
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Toxin neutralization potential of polyclonal anti-HAB antibodies was assessed on RAW 264.7 cell line. Briefly, crude toxins isolated and concentrated from 24 h S. aureus cultures were treated with different dilutions of antisera (1:100, 1:250, 1:500). The effect of toxins on cell viability and morphology was evaluated and the ability of serum antibodies to neutralize the toxins was established by MTT assay and microscopic studies. MTT assay was performed in 96-well cell culture plates (Nunc) (Reddy et al. 2015 (link)). Briefly, cells were cultured at 37 °C with 5% CO2 until ~ 70% confluency. Later cells were incubated with toxin and different serum dilutions in DMEM and incubated for 24 h. Serum from control group (PBS) of mice served as negative control. Spent media were aspirated and 50 μL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrasolium bromide (MTT) in DMEM was added to the wells and incubated for further 4 h at 37 °C. DMSO was added to solubilize the formazan crystals. Absorbance was measured at 570 nm. Toxin neutralization percentage was calculated using the formula: (OD of toxin only) – (OD of toxin + antibody)/(OD of toxin only) – (OD of no toxin) × 100. The morphology of the treated and untreated cells was observed using phase contrast microscopy (Olympus).
7
Mitochondrial Membrane Potential Analysis
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Mitochondrial functionality was assessed by the fluorescent dye Rhodamine 123 (Sigma). Rhodamine 123 is a cationic fluorescent dye that has high affinity with the electrical potential of membranes; thus, it marks active mitochondria in living cells, i.e. the loss of mitochondrial membrane potential is observed as a decrease in the fluorescent signal. Initially, C. tropicalis yeast cells (1 × 104 cells.ml−1) were incubated in Sabouraud broth containing 10 μg.ml−1CaThi, with the final assay volume adjusted to 200 μl. The assay was performed on 96-well cell culture plates (Nunc) at 30°C for 24 h. After incubation with CaThi, C. tropicalis cells were resuspended in the Saboraud medium and incubated with 10 μg.ml−1 Rhodamine 123, with constant orbital agitation at 500 rpm for 2 h and protected from light, and then analyzed by DIC under an optical microscope equipped with a fluorescence filter (excitation wavelength: 506 nm, emission wavelength: 530 nm). Control cells were treated in the same manner, except CaThi was excluded.
8
Dynamic Passivation of 45S5-BG Granules
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45S5-BG granules were subjected to dynamic passivation in DMEM high glucose (Life Technologies, Darmstadt, Germany) for 1 h, 6 h, 24 h, and 72 h respectively. After the designated period of passivation, pH values of the media utilized for the passivation process were analyzed using pure DMEM as control. Conditioned medium was discarded and passivated BG granules as well as non-passivated BG granules (0 h) were added to fresh CCM (89% DMEM high glucose, 10% fetal calf serum (FCS; Life Technologies, Darmstadt, Germany), 1% penicillin/streptomycin (Biochrom, Berlin, Germany)). Medium containing the BG granules was placed in 96-well cell culture plates (Nunc, Roskilde, Denmark) and BMSCs were added achieving a cell density of 2 × 104 cells per cm2 and final BG concentrations of 1, 2.5, and 5 mg/mL. A control group was composed of BMSCs in BG-free CCM while each group consisted of 5 biological replicates. After 1 (D1) and 3 (D3) days of incubation the middle concentration of 2.5 mg/mL was used to evaluate pH values in the CCM and microscopically analyze cell morphology and growth patterns in direct presence of the differently preconditioned BGs. To quantify the influence on cell viability a fluorescence-based assay was performed for all groups as schematically depicted in Figure 1 .
9
Retraction Agent's Effect on Cell Viability
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HGFs were seeded into 96-well cell culture plates (Nunc). After 24 h, the culture medium was replaced with a fresh medium containing the proper amount of the studied retraction agent (the final concentrations of 1 mg/mL and 0.5 mg/mL were obtained). The cells were incubated with the astringents for 5, 10, or 30 min. Additionally, to assess the long-term effect, a 24 h incubation was performed at a concentration of 1 mg/mL. Once it ended, the culture medium with retraction agent was replaced with a fresh DMEM and a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used. Cells were incubated for 90 min with 100 μL of the MTT reagent (Sigma-Aldrich) at 37 °C. Then, formazan crystals were dissolved by adding 100 μL of acidic isopropanol and by mixing. The absorbance was measured at 560 nm using a multiwell plate reader (GloMax Discover Microplate Reader, Promega, Madison, WI, USA). The results are expressed as the percentage of treated cells with altered mitochondrial function in relation to untreated control cells with normal mitochondrial activity, which was considered as 100% cell viability.
10
Retraction Agents Impact on Cell Proliferation
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HGFs were seeded into 96-well cell culture plates (Nunc, Roskilde, Denmark). After 24 h, the culture medium was replaced with a fresh medium containing the proper amount of the studied retraction agent (the final concentration of 1 mg/mL was selected). The cells were incubated with the astringents for 5 min, 10 min, or 24 h. Measurement of bromodeoxyuridine (BrdU) incorporation into newly synthesised DNA strands was performed to detect actively proliferating cells. The BrdU Cell Proliferation Assay Kit (Millipore, Burlington, Massachusetts, USA) was used per the manufacturer’s instructions. The absorbance was measured at 450 nm using a multiwell plate reader (GloMax Discover Microplate Reader, Promega, Madison, WI, USA). The results are expressed as the percentage of treated cells with an altered BrdU incorporation ability compared to untreated control cells with normal ability, which was considered to be 100% cellular proliferation.
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