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In Vitro Techniques

In Vitro Techniques: Investigative procedures performed on materials or biological components outside of a living organism, including cell culture, organ perfusion, and the like.
These techniques are used in basic research and clinical studies to better understand biological processes and diseases as well as to test and develop new medications and diagnostic procedures.
By leveraging innovative AI-powered tools, researchers can optimize their in vitro workflows and ensrue the reproducibility and efficiency of their studies.

Most cited protocols related to «In Vitro Techniques»

1
Preparing ChIP-seq Data and Motif Enrichment Analysis
We use 13 mouse embryonic stem cell (ES) TF ChIP-seq data sets (15 (link)) and the mouse embryonic fibroblast (EF) ChIP-seq data set for NFIC (16 (link)). To prepare the Chen et al. (15 (link)) data sets for use we map the (centers of the) ChIP-seq peaks declared by the authors to the genome, and extract the 500 bp of genomic sequence centered on each peak in FASTA format. To prepare the NFIC data set for use we download the author-defined peaks from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo), (GSM398010_NFI_peaks_wtMEF_300bpwindow_300bpvicinity_range.bed.gz), and use the UCSC genome browser to extract 500bp genomic regions centered 150 bp downstream of each given locus. (The given loci are 150 bp upstream of the centers of the declared peaks.)
For MEA, we use a compendium of motifs consisting of all vertebrate motifs in the JASPAR CORE database (17 (link)) plus all motifs derived for mouse TFs in the UniPROBE database (18 (link)). This compendium contains 532 motifs. We have made no effort to reduce the redundancy of the motif database because we believe doing so is generally unwarranted in central motif enrichment analysis. The statistical power of CMEA is extremely high, so the redundancy has little effect on its ability to detect enriched motifs, and duplicate motifs are often of varying (unknown) quality or were derived using different methods (e.g. in vivo versus in vitro methods), so including them in the analysis can be informative.
Bailey T.L, & Machanick P. (2012). Inferring direct DNA binding from ChIP-seq. Nucleic Acids Research, 40(17), e128.
Publication 2012
Chromatin Immunoprecipitation Sequencing Embryo Fibroblasts Gene Expression Genome In Vitro Techniques Mouse Embryonic Stem Cells Mus Vertebrates
2
Prenatal Toxicant Exposure and Preterm Birth
This study was conducted among pregnant women participating in the “Puerto Rico Testsite for Exploring Contamination Threats (PROTECT)” project, an ongoing prospective birth cohort in the Northern Karst Region of Puerto Rico, which is designed to evaluate the relationship between environmental toxicants and risk of preterm delivery. Study participants were recruited at approximately 14±2 weeks of gestation at seven prenatal clinics and hospitals throughout Northern Puerto Rico during 2010-2012. Women were eligible if they were between the ages of 18 to 40 years, resided in a municipality within the Northern karst region, didn’t use oral contraceptives three months prior to pregnancy or in vitro fertilization as a method of assisted reproductive technology, and were free of known medical/obstetrics complications. Women provided spot urine samples at three separate study visits (20±2 weeks, 24±2 weeks, and 28±2 weeks of gestation). Questionnaires to collect demographic information and data on self-reported product use in the 48 hours preceding urine sample collection were also administered at each visit.
The present analysis reflects the first105 women recruited into the study who had urinary biomarker data as of June 2012. The research protocol was approved by the Ethics and Research Committees of the University of Puerto Rico and participating clinics, the University of Michigan School of Public Health, and Northeastern University. The involvement of the Centers for Disease Control and Prevention (CDC) laboratory was determined not to constitute engagement in human subjects research. The study was described in detail to all participants, and informed consent was obtained prior to study enrollment.
Meeker J.D., Cantonwine D.E., Rivera-González L.O., Ferguson K.K., Mukherjee B., Calafat A.M., Ye X., Anzalota Del Toro L.V., Crespo N., Jiménez-Vélez B., Alshawabkeh A.N, & Cordero J.F. (2013). Distribution, variability and predictors of urinary concentrations of phenols and parabens among pregnant women in Puerto Rico. Environmental science & technology, 47(7), 3439-3447.
Publication 2013
Assisted Reproductive Technologies Biological Markers Birth Cohort Contraceptives, Oral Fertilization Fertilization in Vitro Homo sapiens In Vitro Techniques Pregnancy Pregnant Women Premature Birth Urine Urine Specimen Collection Woman
3
Prenatal Phthalate Exposure in Puerto Rico

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Publication 2013
Assisted Reproductive Technologies Birth Cohort Contraceptives, Oral Fertilization Fertilization in Vitro Homo sapiens In Vitro Techniques Mothers phthalate Pregnancy Pregnant Women Premature Birth Urine
4
Recombinant PCR for Genome-Wide Gene Replacement
We developed a recombinant PCR method for in vitro creation of linear constructs for the replacement of every protein-coding gene in the S. sanguinis SK36 genome (Figure S1A). Based on the complete S. sanguinis SK36 genome sequence24 (link), three sets of primers (F1/R1, F2/R2 and F3/R3) were designed to amplify the S. sanguinis sequence upstream from each targeted gene, the aphA-3 gene, encoding Kmr45 (link) and the S. sanguinis sequence downstream from each targeted gene, respectively.
For most of the mutagenized genes, the R1 and F3 primers were designed to delete the coding region from 6 bp after the start codon to 30 bp before the stop codon. Stop codons were inserted in all three frames to prevent fusion of the N-terminus of the targeted open reading frame with the Kmr protein. The last 30 bp were retained to preserve potential ribosomal binding sites used by adjacent downstream genes. The upstream retained region was extended from 6 bp to 100 bp when two neighboring genes were located head-to-head in opposite orientation to prevent deletion of potential promoters for flanking genes. Primers R1 and F3 contained 25-nt sequences that are complementary with the antibiotic selection cassette at their 5' ends. The P1, P2, and various T1 primers were designed for sequencing to confirm mutants. The sequence of every primer is documented in Table S1.
Three PCR amplicons were created using F1/R1, F2/R2 and F3/R3. All PCR reactions were performed at 94°C for 1 min, and 30 cycles of 94°C for 30 sec, 54°C for 30 sec and 68°C for 1.5 min. After DNA purification by PureLink 96 PCR purification kits (Invitrogen), the three PCR amplicons were combined in equal amounts in one tube and amplified again using the F1 and R3 primers to obtain the final linear recombinant PCR amplicon. Conditions were 94°C for 2 min, 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 68°C for 3.5 min, and finally 68°C for 4 min. High-fidelity Platinum® Taq DNA polymerase (Invitrogen) was used in all reactions.
Xu P., Ge X., Chen L., Wang X., Dou Y., Xu J.Z., Patel J.R., Stone V., Trinh M., Evans K., Kitten T., Bonchev D, & Buck G.A. (2011). Genome-wide essential gene identification in Streptococcus sanguinis. Scientific Reports, 1, 125.
Publication 2011
Antibiotics Binding Sites Codon, Initiator Codon, Terminator Deletion Mutation Gene Products, Protein Genes Genome Head In Vitro Techniques Oligonucleotide Primers Platinum Proteins Reading Frames Ribosomes Taq Polymerase
5
Characterization and In Vitro Evaluation of Microsponge-Based Gels

Appearance

The formulated gels were examined visually for their color, appearance, and consistency.

Determination of pH

The pH of the formulation was determined by using a digital pH meter (HANNA 211).

Determination of viscosity

The viscosity of the formulation was studied by a digital viscometer (Rotary viscometer STS-2011) using spindle R7 at 25 ± 10 °C.

Determination of drug content

One gram of formulation was diluted with 100 mL of ethanol 95% with proper mixing. The solution was filtered and was analysed for drug content by HPLC method with proper dilution of the sample.

In vitro drug release studies

In vitro release studies were performed using cellulose nitrate membrane. For this experiment, a vertical Franz diffusion cell with a surface area of 2.54 cm2 and a reservoir capacity of 9.5 mL was used. The membrane was placed between the two halves of the diffusion cell. The receptor compartment contained a mixture of water and ethanol (50:50, v/v), and its temperature was maintained at 32 ± 0.1 °C and stirred continuously using a magnetic stirrer. Each formulation weighing 0.5 g of microsponge based gel was placed on the donor side. A total of 2 mL of the sample was withdrawn from the receptor compartment at definite time intervals and replaced with an equal volume of fresh receptor fluid. The aliquots were suitably diluted with the receptor medium and analysed by HPLC method14 (link)–16 (link).

In vitro drug release kinetics

To investigate the release mechanism of CLN-free base from the microsponge loaded gels, the release data was analysed using zero order, first order, Higuchi, Hixson-Crowell, and Korsmeyer-Peppas.

Stability study

Optimized batches of CLN microsponge gels were monitored for up to 6 months at 40 ± 2°/75 ± 5% RH as per ICH guidelines17 . At the interval of 1, 2, 3, 4, 5, and 6 months, samples were withdrawn and analysed to determine changes in appearance, pH, viscosity and drug content, and drug release14 (link).

KHATTAB A, & Nattouf A. (2021). Optimization of entrapment efficiency and release of clindamycin in microsponge based gel. Scientific Reports, 11, 23345.
Publication 2021
Cells Diffusion Drug Liberation Ethanol Fingers Gels High-Performance Liquid Chromatographies In Vitro Techniques Nitrocellulose Pharmaceutical Preparations Place Cells Technique, Dilution Tissue, Membrane Tissue Donors Viscosity

Most recents protocols related to «In Vitro Techniques»

1
Rice Nitrogen and Antioxidant Enzymes
Rice plants with consistent growth and development (sampling by population mean stem number) were selected at the panicle initiation and heading stage and 20 days after heading. The flag leaves (the first fully expanded leaf under the heart leaf before heading) were sampled and frozen in liquid nitrogen and stored at -80°C to analyse their nitrogen metabolic enzymes and antioxidant enzymes. Nitrate reductase (NR) activity was determined according to the in vitro method described by Li et al. (2000) . The enzyme activity was expressed in the number of micrograms of NaNO2 produced per gram of sample per hour (μg/(h·g) (calculated as NaNO2, the same below). Glutamine synthetase (GS) was determined according to the method described by Wang et al. (2005) . Glutamate dehydrogenase (GDH) was determined according to the method described by Masclaux et al. (2000) (link). The superoxide dismutase (SOD) activity and peroxidase (POD) activity were measured according to the method described by Qiu et al. (2010) (link). Catalase (CAT) activity was measured using the UV absorption method (Zeng et al., 1991 ).
Zhao L., Tang Q., Song Z., Yin Y., Wang G, & Li Y. (2023). Increasing the yield of drip-irrigated rice by improving photosynthetic performance and enhancing nitrogen metabolism through optimizing water and nitrogen management. Frontiers in Plant Science, 14, 1075625.
Publication 2023
Antioxidants Catalase enzyme activity Enzymes Freezing Glutamate-Ammonia Ligase Glutamate Dehydrogenase Heart In Vitro Techniques Nitrate Reductase Nitrogen Oryza sativa Peroxidase Plant Development Plant Leaves Stem, Plant Superoxide Dismutase
2
Affinity-Based Protein Pulldown Assay
The pulldown experiment is an in vitro technique used to detect physical interactions between two or more proteins and is a valuable tool for confirming predicted protein-protein interactions or identifying new interaction partners. ABPP is a powerful method that can help to identify the cellular targets of bioactive molecules. In general, the probe molecule is designed to insert a terminal acetylene into the bioactive parent molecule to facilitate Cu(I)-catalysed click reactions with azide affinity markers (Darabedian et al., 2018 (link); Li et al., 2019 (link)). The probe used in our experiment is a molecule formed by inserting a terminal acetylene into the molecule of NBP, so it can pulldown the binding protein via ABPP. We dissolved NBP or probe in DMSO and added 100 μM of NBP or probe to the medium of treated bEnd.3 cells immediately after reoxidation. The treated cells were collected 18 h after reoxygenation and placed in RIPA lysis buffer (Beyotime, China) to extract the proteins. The protein solution was incubated at 20–25 °C for 1 h with 100 μM Biotin azide (Sigma-Aldrich, United States), 1.0 mM CuSO4 (Sigma-Aldrich, United States), 100 μM THTPA (Sigma-Aldrich, United States), and 100 μM NaVc (Sigma-Aldrich, United States) for the “click” reaction. Subsequently, 900 μL of buffer (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% SDS) and 40 μL of streptavidin-sepharose beads (Sigma-Aldrich, United States) were added, rotating continuously overnight at 4°C. After washing the beads with buffer three times, the eluted protein was separated via SDS-PAGE. Afterward, the eluted protein was stained using a Fast Silver Stain Kit (Beyotime, China) or transferred to nitrocellulose membranes (Millipore, United States) for Western blot analysis.
Jia J., Deng J., Jin H., Yang J., Nan D., Yu Z., Yu W., Shen Z., Lu Y., Liu R., Wang Z., Qu X., Qiu D., Yang Z, & Huang Y. (2023). Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury. Frontiers in Pharmacology, 14, 1084564.
Publication 2023
Acetylene Azides Binding Proteins biotin 1 bropirimine Buffers Cells Decompression Sickness In Vitro Techniques Nitrocellulose Parent Physical Examination Proteins Radioimmunoprecipitation Assay SDS-PAGE Silver Sodium Chloride Stains Staphylococcal Protein A streptavidin-agarose Sulfoxide, Dimethyl Tissue, Membrane Tromethamine Western Blot
3
Optimized Oocyte Handling for ICSI
Warmed oocytes were cultured for three hours before ICSI. Fresh oocytes were denudated immediately following oocyte retrieval. In vitro insemination procedures were performed 38 to 39 h post triggering for fresh oocytes, with exceptions in male factor, in which ICSI was performed instead. In addition, assisted hatching was performed to improve embryo capacity to implant.
Lee K.S., Lin M.H., Hwu Y.M., Yang J.H, & Lee R.K. (2023). The live birth rate of vitrified oocyte accumulation for managing diminished ovarian reserve: a retrospective cohort study. Journal of Ovarian Research, 16, 49.
Publication 2023
Embryo Insemination In Vitro Techniques Males Oocyte Retrieval Oocytes Sperm Injections, Intracytoplasmic
4
Establishing Drug-Resistant Cell Lines
Human Intestinal Epithelial Cells (HIEC) and Human colorectal adenocarcinoma cells (HCT116), purchased from American Type Culture Collection, were cultured in 1640 medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin, and the medium was changed every 2 days. The cell line of HCT116 was obtained from American Type Culture Collection (USA). While, the OX-resisted cell line of HCT116/DR was obtained from Shanghai Meixuan Biotechnology Co., Ltd., which was establishment on HCT116 (American Type Culture Collection, USA) by a drug-induced method with gradually increased OX concentration. The basic principle of building drug-resistant cell lines in vitro by increasing drug concentration method is that long-term contact with low-dose drugs causes changes in the drug chemical process of the cell itself, and the appearance of Pgp glycoprotein (P-glycoprotein) on the cell membrane makes the cell gradually tolerant to drugs [37 (link), 38 (link)].
Pei M., Liu K., Qu X., Wang K., Chen Q., Zhang Y., Wang X., Wang Z., Li X., Chen F., Qin H, & Zhang Y. (2023). Enzyme-catalyzed synthesis of selenium-doped manganese phosphate for synergistic therapy of drug-resistant colorectal cancer. Journal of Nanobiotechnology, 21, 72.
Publication 2023
Adenocarcinoma Cell Lines Cells Epithelial Cells Fetal Bovine Serum Glycoproteins Homo sapiens Intestines In Vitro Techniques P-Glycoprotein Penicillins Pharmaceutical Preparations Physiology, Cell Plasma Membrane Streptomycin
5
Statistical Methods for Biological Research
Sample size was chosen considering at least three independent experiment for trivial in vitro technics with cell lines (RT-qPCR, western blot). For chromatin immunoprecipitation we performed independent experiment with at least two pairs of primers (up to four). For animal studies we studied as many littermates as possible for 1 years (n = 24; 28 and 12).
Statistical analysis was performed using Prism v 6.0 (GraphPad software Inc., San Diego, CA) or R statistical v 3.3.1 software. For analysis of different measurements, a normality test was conducted, when the number of samples was sufficient. Variance was assessed to test for possible statistical analysis. For samples following a normal Gaussian distribution, a Student t-test was applied, either paired or unpaired, depending on the experimental data. When samples did not pass the normality test, a non-parametric test was applied (Mann–Whitney for unpaired samples and Wilcox on signed-rank test for paired samples). The correlation between the expression and methylation of Notch3 was done using a Cox test. The statistics on IHC of normal tissues compared to tumor tissues of patients with respect to cell localizations was done using a Chi-squared test of conformity. Differences between groups of the survival analysis were tested by log-rank tests. *p < 0.05; **p < 0.01; ***p < 0.001.
Brahim S., Negulescu A.M., Geneste C., Schott T., Lin S., Morel L.O., Rama N., Gadot N., Treilleux I., Mehlen P, & Meurette O. (2023). Notch3 regulates Mybl2 via HeyL to limit proliferation and tumor initiation in breast cancer. Cell Death & Disease, 14(2), 171.
Publication 2023
Animals Cell Lines Cells Immunoprecipitation, Chromatin In Vitro Techniques Methylation Neoplasms NOTCH3 protein, human Oligonucleotide Primers Patients prisma Student Tissues Western Blotting

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1
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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Penicillin is a type of antibacterial drug that is widely used in medical and laboratory settings. It is a naturally occurring substance produced by certain fungi, and it is effective against a variety of bacterial infections. Penicillin works by inhibiting the growth and reproduction of bacteria, making it a valuable tool for researchers and medical professionals.
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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Epidermal growth factor (egf) by Thermo Fisher Scientific
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EGF is a lab equipment product from Thermo Fisher Scientific. It is a recombinant human Epidermal Growth Factor (EGF) protein. EGF is a growth factor that plays a role in cell proliferation and differentiation.
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More about "In Vitro Techniques"

In vitro techniques encompass a wide range of investigative procedures performed on materials or biological components outside of a living organism.
These methods are crucial in basic research and clinical studies, enabling researchers to better understand biological processes, diseases, and test new medications and diagnostic tools.
Some common in vitro techniques include cell culture, organ perfusion, and a variety of other lab-based experiments.
Leveraging innovative AI-powered tools, such as PubCompare.ai, can help optimize in vitro workflows and ensure the reproducibility and efficiency of these studies.
PubCompare.ai allows researchers to locate protocols from literature, pre-prints, and patents, and compare techniques and products to identify the most effective approaches.
In the realm of in vitro research, various materials and reagents play a crucial role.
Fetal bovine serum (FBS) is a commonly used supplement in cell culture media, providing essential growth factors and nutrients.
Antibiotics like penicillin and streptomycin are often added to cell culture systems to prevent bacterial contamination.
The Adeno-X Rapid Titer Kit, for example, is a tool used to quantify adenovirus titers in vitro.
Analytical techniques, such as UV spectroscopy, are employed to characterize and quantify biomolecules in in vitro experiments.
Specialized cell culture flasks, coated with extracellular matrix (ECM) proteins like ECM625, can provide a more physiologically relevant microenvironment for cell growth and differentiation.
Incorporating epidermal growth factor (EGF) into in vitro models can stimulate cell proliferation and migration, while the use of Dulbecco's Modified Eagle Medium (DMEM) provides a nutrient-rich environment for cell culture.
By leveraging these tools and techniques, researchers can optimize their in vitro workflows, ensure reproducibility, and advance our understanding of biological systems and disease mechanisms.
The future of in vitro research is being shaped by the integration of innovative AI-powered tools, such as PubCompare.ai, which empower scientists to explore new frontiers in a more efficient and effective manner.