Cell Proliferation
This biological phenomenon is essential for growth, development, tissue repair, and the maintenance of homeostasis in multicellular organisms.
Factors that regulate cell proliferation include growth factors, cytokines, cell-cell and cell-matrix interactions, and the cell cycle machinery.
Dysregulation of cell proliferation can lead to pathological conditions such as cancer, autoimmune disorders, and fibrotic diseases.
Researchers studying cell proliferation utilize a variety of techniques, including cell culture assays, flow cytometry, and molecular biology methods.
Optimizing experimental protocols and leveraging innovative technologies, such as AI-driven comparisons of proliferation data, can boost research productivity and reproducibility in this important field of study.
Most cited protocols related to «Cell Proliferation»
Cytotoxicity was measured using the XTT cell proliferation Kit II and MPBA. The method described by Zheng et al. [8 (link)] was used to perform the assay. Both cell lines were seeded in a 96-well microtitre plate at a concentration of 1 × 105 cells/mL. Cells were allowed to attach for 24 hrs at 37°C and 5% CO2. The cells were exposed to the positive drug control Actinomycin D (Sigma-Aldrich, South Africa) with concentrations ranging between 0.5 µg/mL and 0.002 µg/mL. The microtitre plate was incubated for further 72 hrs and thereafter 50 µL XTT was added to a final concentration of 0.3 mg/mL to one set of plates and 20 µL PrestoBlue was added to another set of plates. The plates were incubated for further 2 hrs where after the absorbance of the colour complex was read at 490 nm with a reference wavelength set at 690 nm for XTT and at 570 nm with a reference wavelength set at 600 nm for PrestoBlue, using a BIO-TEK Power-Wave XS multiwell plate reader.
Cell proliferation and cytotoxicity experiments were performed using modified 16-well plates (E-plate, Roche Diagnostics GmbH, Mannheim, Germany). Microelectrodes were attached at the bottom of the wells for impedance-based detection of attachment, spreading and proliferation of the cells. Initially, 100 µL of cell-free growth medium (10% FBS) was added to the wells. After leaving the devices at room temperature for 30 min, the background impedance for each well was measured. Cells were harvested from exponential phase cultures by a standardized detachment procedure using 0.05% Trypsin-EDTA (Invitrogen NV/SA, Merelbeke, Belgium) and counted automatically with a Scepter 2.0 device (Merck Millipore SA/NV, Overijse, Belgium), Fifty µL of the cell suspension was seeded into the wells (20, 40, 80, 100, 200, 400 and 800 cells/well for proliferation, 1000 cells/well for cytotoxicity experiments). The cell concentrations of 20, 100, 200 and 400 cells/well were considered for correlation with the SRB method described below. After leaving the plates at room temperature for 30 min to allow cell attachment, in accordance with the manufacturer's guidelines, they were locked in the RTCA DP device in the incubator and the impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value (CI). Water was added to the space surrounding the wells of the E-plate to avoid interference from evaporation. For proliferation assays, the cells were incubated during ten days in growth medium (10% FBS) and CI was monitored every 15 min during the first six hours, and every hour for the rest of the period. Two replicates of each cell concentration were used in each test. For cytotoxicity experiments, CI of each well was automatically monitored with the xCELLigence system every 15 min during the overnight recovery period. Twenty-four hours after cell seeding, cells were treated during a period of 72 hours with paclitaxel (0, 1, 2, 5, 10, 20, 50 and 100 nM) dissolved in phosphate buffered saline (PBS). PBS alone was added to control wells. Each concentration was tested in duplicate within the same experiment. CI was monitored every 15 min during the experiment. Three days after the start of treatment with paclitaxel, CI measurement was ended.
Most recents protocols related to «Cell Proliferation»
Example 7
The MTT Cell Proliferation assay determines cell survival following apple stem cell extract treatment. The purpose was to evaluate the potential anti-tumor activity of apple stem cell extracts as well as to evaluate the dose-dependent cell cytotoxicity.
Principle: Treated cells are exposed to 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). MTT enters living cells and passes into the mitochondria where it is reduced by mitochondrial succinate dehydrogenase to an insoluble, colored (dark purple) formazan product. The cells are then solubilized with DMSO and the released, solubilized formazan is measured spectrophotometrically. The MTT assay measures cell viability based on the generation of reducing equivalents. Reduction of MTT only occurs in metabolically active cells, so the level of activity is a measure of the viability of the cells. The percentage cell viability is calculated against untreated cells.
Method: A549 and NCI-H520 lung cancer cell lines and L132 lung epithelial cell line were used to determine the plant stem cell treatment tumor-specific cytotoxicity. The cell lines were maintained in Minimal Essential Media supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a 5% CO2 at 37 Celsius. Cells were seeded at 5×103 cells/well in 96-well plates and incubated for 48 hours. Triplicates of eight concentrations of the apple stem cell extract were added to the media and cells were incubated for 24 hours. This was followed by removal of media and subsequent washing with the phosphate saline solution. Cell proliferation was measured using the MTT Cell Proliferation Kit I (Boehringer Mannheim, Indianapolis, IN) New medium containing 50 μl of MTT solution (5 mg/ml) was added to each well and cultures were incubated a further 4 hours. Following this incubation, DMSO was added and the cell viability was determined by the absorbance at 570 nm by a microplate reader.
In order to determine the effectiveness of apple stem cell extracts as an anti-tumor biological agent, an MTT assay was carried out and IC50 values were calculated. IC50 is the half maximal inhibitory function concentration of a drug or compound required to inhibit a biological process. The measured process is cell death.
Results: ASC-Treated Human Lung Adenocarcinoma Cell Line A549.
Results: ASC-Treated Human Squamous Carcinoma Cell Line NCI-H520.
Results: ASC-treated Lung Epithelial Cell Line L132.
Summary Results: Cytotoxicity of Apple Stem Cell Extracts.
Apple stem cell extracts killed lung cancer cells lines A549 and NCI-H520 at relatively low doses: IC50s were 12.58 and 10.21 μg/ml respectively as compared to 127.46 μg/ml for the lung epithelial cell line L132. Near complete anti-tumor activity was seen at a dose of 250 μg/ml in both the lung cancer cell lines. This same dose spared more than one half of the L132 cells. See Tables 7-10. The data revealed that apple stem cell extract is cytotoxic to lung cancer cells while sparing lung epithelial cells.
Example 9
The experiment of Example 7 was repeated substituting other plant materials for ASC. Plant stem cell materials included Dandelion Root Extract (DRE), Aloe Vera Juice (AVJ), Apple Fiber Powder (AFP), Ginkgo Leaf Extract (GLE), Lingonberry Stem Cells (LSC), Orchid Stem Cells (OSC) as described in Examples 1 and 2. The concentrations of plant materials used were nominally 250, 100, 50, 25, 6.25, 3.125, 1.562, and 0.781 μg/mL. These materials were tested only for cells the human lung epithelial cell line L132 (as a proxy for normal epithelial cells) and for cells of the human lung adenocarcinoma cell line A549 (as a proxy for lung cancer cells).
A549 cells lung cancer cell line cytotoxicity results for each of the treatment materials.
DRE-Treated Lung Cancer Cell Line A549 Cells.
AVJ-Treated Lung Cancer Cell line A549 Cells.
AFP-Treated Lung Cancer Cell line A549 Cells.
GLE-treated Lung Cancer Cell line A549 Cells.
LSC-treated lung cancer cell lines A549 cells.
OSC-treated Lung Cancer Cell line A549 Cells.
L132 cells (“normal” lung epithelial cell line) cytotoxicity results for each of the treatment materials.
DRE-Treated Lung Epithelial Cell Line L132 cells.
AVJ-Treated Lung Epithelial Cell Line L132 cells.
AFP-Treated Lung Epithelial Cell Line L132 cells.
GLE-Treated Lung Epithelial Cell Line L132 cells.
LSC-Treated Lung Epithelial Cell Line L132 cells.
OSC-Treated Lung Epithelial Cell Line L132 cells.
Calculated values.
Example 7
Impact of IL-2 signalling on Teff responses is characterised in a T cell activation assay, in which intracellular granzyme B (GrB) upregulation and proliferation are examined. Previously frozen primary human Pan T cells (Stemcell Technologies) are labelled with eFluor450 cell proliferation dye (Invitrogen) according to manufacturer's recommendation, and added to 96-U-bottom well plates at 1×105 cells/well in RPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mM L-Glutamine (Life Technologies) and 10,000 U/ml Pen-Strep (Sigma). The cells are then treated with 10 μg/ml anti-CD25 antibodies or control antibodies followed by Human T-Activator CD3/CD28 (20:1 cell to bead ratio; Gibco) and incubated for 72 hrs in a 37° C., 5% CO2 humidified incubator. To assess T cell activation, cells are stained with the eBioscience Fixable Viability Dye efluor780 (Invitrogen), followed by fluorochrome labelled antibodies for surface T cell markers (CD3-PerCP-Cy5.5 clone UCHT1 Biolegend, CD4-BV510 clone SK3 BD Bioscience, CD8-Alexa Fluor 700 clone RPA-T8 Invitrogen, CD45RA-PE-Cy7 clone HI100 Invitrogen, CD25-BUV737 clone 2A3 BD Bioscience) and then fixed and permeabilized with the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Invitrogen) before staining for intracellular GrB and intranuclear FoxP3 (Granzyme B-PE clone GB11 BD Bioscience, FoxP3-APC clone 236A/E7). Samples are acquired on the Fortessa LSR X20 Flow Cytometer (BD Bioscience) and analysed using the BD FACSDIVA software. Doublets are excluded using FCS-H versus FCS-A, and lymphocytes defined using SSC-A versus FCS-A parameters. CD4+ and CD8+ T cell subsets gated from the live CD3+ lymphocytes are assessed using a GrB-PE-A versus proliferation eFluor450-A plot. Results are presented as percentage of proliferating GrB positive cells from the whole CD4+ T cell population. Graphs and statistical analysis is performed using GraphPad Prism v7. (results not shown)
Example 8
Cell adhesion was also evaluated by means of in vitro scratch wound-healing assay. HDPSCs cells were analyzed by difference in staining with phalloidin (cell nucleus) and DAPI to visualize actin cytoskeleton.
Cell adhesion results showed excellent interaction and adhesion between neighboring cells in the presence of bioceramic composition. The Bioceramic composition sealer (CB5) and Bioceramic composition repair (CB6), showed a gradual increase in growth over time, an extended morphology and a high content of F-Actin (cell microfilamen), reaching confluence after 72 hours of culture.
The analysis of cell proliferation (via cell viability study), apoptosis, cell adhesion and morphology (via cell adhesion study) and migration (via cell migration study) showed very positive results, indicating that the proposed bioceramic composition induces the odonto/osteogenic mineralization and differentiation process in the presence of tooth-specific human stem cells (hDPSCs pulp). While a market resin sealer was also used in the comparative studies, however, all results were not satisfactory for this product.
Example 3
Lung cancer cell line A549 and squamous cell carcinoma cell line H10 expressing inducible SEQ ID NO: 1-HA vector were established as described previously. SEQ ID NO: 1 expression was detected by qPCR (
To evaluate the effects of SEQ ID NO: 1 on proliferation, A549 and H10 cells transduced with SEQ ID NO: 1-HA vector or control vector were monitored for 14 days. Growth curves show that cells overexpressing micropeptide SEQ ID NO: 1 have a consistently lower growth rate compared to the control (
Example 6
The Effect of ARTS Mimetic Small Molecule A4 on Premalignant Cells
Acini-like organoids forms hollow lumen after 10 to 12 days in 3D culture system and remain hollow thereafter (Muthuswamy et al., 2001). Plasmids introduced by transient cell transfection are only expressed for a limited period of time, as they are not integrated into the genome and therefore may be lost by environmental factors and cell division. Therefore, the inventors next examined whether introduction of small-molecules may mimic ARTS function, specifically in inducing lumen formation and reversion of pre-malignant cells to a normal-like phenotype. The inventors thus tested initially the effect of the ARTS mimetic small molecule A4 on induction of apoptosis in 2D culture.
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More about "Cell Proliferation"
Cell proliferation is a fundamental biological process essential for growth, development, tissue repair, and homeostasis in multicellular organisms.
It involves the division and increase in the number of cells, regulated by various factors such as growth factors, cytokines, cell-cell and cell-matrix interactions, and the cell cycle machinery.
Dysregulation of cell proliferation can lead to pathological conditions like cancer, autoimmune disorders, and fibrotic diseases.
Researchers utilize a variety of techniques, including cell culture assays (e.g., CellTiter 96® AQueous One Solution, Cell Counting Kit-8), flow cytometry, and molecular biology methods to study cell proliferation.
Optimizing experimental protocols and leveraging innovative technologies, such as AI-driven comparisons of proliferation data (e.g., PubCompare.ai), can boost research productivity and reproducibility in this important field of study.
By understanding the key concepts, terminology, and available tools, researchers can design more effective cell proliferation experiments and advance our knowledge in this critical area of biology and medicine.