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Tissue Culture Techniques

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Most cited protocols related to «Tissue Culture Techniques»

1
Isolation and Culture of Vascular Cells
Cited 4 times
Cells were isolated from the blood vessels in rat, mouse and human. Human carotid arteries were obtained from National Disease Research Interchange (Philadelphia, PA). The cells isolation methods were described previously 56 (link). Briefly, the tissue segments were washed three times with phosphate buffered saline (PBS) supplemented with 1% penicillin/streptomycin (P/S). The surrounding connective tissues and adventitia were dissected away under a dissecting microscope. Endothelium was removed by scraping off the cell layer on the luminal surface with sterile scalpel blades. For tissue explant culture method, the tunica media was cut into mm-size and placed onto the surface coated with 1% CellStart (Invitrogen Corp.) in 6-well plates. The cells were cultured in DMEM with 10% FBS (Thermo Fisher Scientific Inc.), or in DMEM with 2% CEE (MP Biomedical, Inc.), 1% FBS, 1% N2 (Invitrogen Corp.), 2% B27 (Invitrogen Corp.), 100 nM retinoic acid (RA) (Sigma-Aldrich, Inc.), 50 nM 2-mercaptoethanol (2ME) (Sigma-Aldrich, Inc.), 1% P/S and 20 ng/ml bFGF (R&D Systems, Inc.) (Maintenance medium). For enzymatic digestion methods, tissues were incubated with 3 mg/ml type II collagenase (Sigma-Aldrich Inc.) in DMEM with a 1/5 (w/v) ratio of tissue (g) to enzyme solution (ml). After incubation at 37°C for 30 min, the same volume of 1 mg/ml elastase (Sigma-Aldrich, Inc.) solution was added to the solution containing the tissue and collagenase. The tissues were incubated for another 1–2 hours until all the tissues were digested. Cells were then seeded onto CellStart-coated dishes and maintained at 37°C in an incubator with 5% CO2.
Tang Z., Wang A., Yuan F., Yan Z., Liu B., Chu J.S., Helms J.A, & Li S. (2012). Differentiation of Multipotent Vascular Stem Cells Contributes to Vascular Diseases. Nature communications, 3, 875.
Publication 2012
2-Mercaptoethanol Adventitia Blood Vessel Carotid Arteries Cells Cell Separation Collagenase Connective Tissue Digestion Endothelium Enzymes Homo sapiens Hyperostosis, Diffuse Idiopathic Skeletal Matrix Metalloproteinase 13 Microscopy Mus Pancreatic Elastase Penicillins Phenobarbital Phosphates Saline Solution Sterility, Reproductive Streptomycin Suby's G solution Tissue Culture Techniques Tissues Tretinoin Tunica Media Type II Mucolipidosis
2
Isolation and Culture of Vascular Cells
Cited 4 times
Cells were isolated from the blood vessels in rat, mouse and human. Human carotid arteries were obtained from National Disease Research Interchange (Philadelphia, PA). The cells isolation methods were described previously 56 (link). Briefly, the tissue segments were washed three times with phosphate buffered saline (PBS) supplemented with 1% penicillin/streptomycin (P/S). The surrounding connective tissues and adventitia were dissected away under a dissecting microscope. Endothelium was removed by scraping off the cell layer on the luminal surface with sterile scalpel blades. For tissue explant culture method, the tunica media was cut into mm-size and placed onto the surface coated with 1% CellStart (Invitrogen Corp.) in 6-well plates. The cells were cultured in DMEM with 10% FBS (Thermo Fisher Scientific Inc.), or in DMEM with 2% CEE (MP Biomedical, Inc.), 1% FBS, 1% N2 (Invitrogen Corp.), 2% B27 (Invitrogen Corp.), 100 nM retinoic acid (RA) (Sigma-Aldrich, Inc.), 50 nM 2-mercaptoethanol (2ME) (Sigma-Aldrich, Inc.), 1% P/S and 20 ng/ml bFGF (R&D Systems, Inc.) (Maintenance medium). For enzymatic digestion methods, tissues were incubated with 3 mg/ml type II collagenase (Sigma-Aldrich Inc.) in DMEM with a 1/5 (w/v) ratio of tissue (g) to enzyme solution (ml). After incubation at 37°C for 30 min, the same volume of 1 mg/ml elastase (Sigma-Aldrich, Inc.) solution was added to the solution containing the tissue and collagenase. The tissues were incubated for another 1–2 hours until all the tissues were digested. Cells were then seeded onto CellStart-coated dishes and maintained at 37°C in an incubator with 5% CO2.
Tang Z., Wang A., Yuan F., Yan Z., Liu B., Chu J.S., Helms J.A, & Li S. (2012). Differentiation of Multipotent Vascular Stem Cells Contributes to Vascular Diseases. Nature communications, 3, 875.
Publication 2012
2-Mercaptoethanol Adventitia Blood Vessel Carotid Arteries Cells Cell Separation Collagenase Connective Tissue Digestion Endothelium Enzymes Homo sapiens Hyperostosis, Diffuse Idiopathic Skeletal Matrix Metalloproteinase 13 Microscopy Mus Pancreatic Elastase Penicillins Phenobarbital Phosphates Saline Solution Sterility, Reproductive Streptomycin Suby's G solution Tissue Culture Techniques Tissues Tretinoin Tunica Media Type II Mucolipidosis
3
Placental Mesenchymal Stromal Cells Isolation
Cited 3 times
We selected 35 placentas that met the requirements (maternal age less than 35 years, negative for infectious diseases, gestational age more than 35 weeks, regular birth examination, and no disease that interferes with placental vigor). Written informed consent was obtained from the donors. The study protocols were reviewed and approved by the Zhujiang Hospital review board and ethics committee of Zhujiang Hospital of Southern Medical University (approval number: 2019-KY-015-02). Thirty samples were treated with the tissue explant method and enzymatic digestion. Placental chorionic vascular MSCs were derived from five other samples after treatment with the umbilical vein perfusion method. The efficiency of different methods for isolation and culture of MSCs from the same placental parts was statistically analyzed, and the average passage time and yield (the number of primary MSCs that could be isolated from tissues of the same weight) were calculated. We selected MSCs from the same individuals (who provided placentas from which UC-MSCs were obtained by the tissue block culture method, and placental MSCs were obtained by enzymatic digestion) for biological characteristics analysis to avoid individual differences. Cells were cultured in serum-free media (Haoyang sc-82,013-G www.tbdscience.com) to minimize the individual differences associated with culture methods, which can impact results.
Yi X., Chen F., Liu F., Peng Q., Li Y., Li S., Du J., Gao Y, & Wang Y. (2020). Comparative separation methods and biological characteristics of human placental and umbilical cord mesenchymal stem cells in serum-free culture conditions. Stem Cell Research & Therapy, 11, 183.
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Publication 2020
Biopharmaceuticals Blood Vessel Cells Chorion Communicable Diseases Culture Media, Serum-Free Culture Techniques Digestion Donors Enzymes Ethics Committees, Clinical Genetic Testing Gestational Age isolation Perfusion Placenta Specimen Handling Tissue Culture Techniques Tissues Umbilical Vein
4
Evaluating SARS-CoV-2 Inhibitors in Vero E6 Cells
Cited 3 times
2,000 Vero E6 cells were seeded into 96-well plates in DMEM (10% FBS) and incubated for 24 h at 37C, 5% CO2. Vero E6 cells used were purchased from ATCC and thus authenticated (VERO C1008 [Vero 76, clone E6, Vero E6] (ATCC® CRL-1586™); tested negative for mycoplasma contamination prior to commencement). Two hours before infection, the medium was replaced with 100ul of DMEM (2% FBS) containing the compound of interest at concentrations 50% greater than those indicated, including a DMSO control. The Vero E6 cell line used in this study is a kidney cell line; therefore, we cannot exclude that lung cells yield different results for some inhibitors (also see Methods Institut Pasteur). Plates were then transferred into the BSL3 facility and 100 PFU (MOI 0.025) was added in 50ul of DMEM (2% FBS), bringing the final compound concentration to those indicated. Plates were then incubated for 48 h at 37C. After infection, supernatants were removed and cells were fixed with 4% formaldehyde for 24 hours prior to being removed from the BSL3 facility. The cells were then immunostained for the viral NP protein (anti-sera produced in the Garcia-Sastre lab; 1:10,000) with a DAPI counterstain. Infected cells (488nM) and total cells (DAPI) were quantified using the Celigo (Nexcelcom) imaging cytometer. Infectivity is measured by the accumulation of viral NP protein in the nucleus of the Vero E6 cells (fluorescence accumulation). Percent infection was quantified as ((Infected cells/Total cells) - Background) *100 and the DMSO control was then set to 100% infection for analysis. The IC50 and IC90 for each experiment were determined using the Prism (GraphPad Software) software. For select inhibitors, infected supernatants were assayed for infectious viral titer using the Median Tissue Culture Infectious Dose (TCID)50 method. For this, infectious supernatants were collected at 48h post infection and frozen at −80°C until later use. Infectious titers were quantified by limiting dilution titration on Vero E6 cells. Briefly, Vero E6 cells were seeded in 96-well plates at 20,000 cells/well. The next day, SARS-CoV-2-containing supernatant was applied at serial 10-fold dilutions ranging from 10−1 to 10−6 and, after 5 days, viral CPE was detected by staining cell monolayers with crystal violet. TCID50/mL were calculated using the method of Reed and Muench. Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer’s instructions. Cytotoxicity was performed in uninfected VeroE6 cells with same compound dilutions and concurrent with viral replication assay. All assays were performed in biologically independent triplicates.
Gordon D.E., Jang G.M., Bouhaddou M., Xu J., Obernier K., White K.M., O’Meara M.J., Rezelj V.V., Guo J.Z., Swaney D.L., Tummino T.A., Huettenhain R., Kaake R.M., Richards A.L., Tutuncuoglu B., Foussard H., Batra J., Haas K., Modak M., Kim M., Haas P., Polacco B.J., Braberg H., Fabius J.M., Eckhardt M., Soucheray M., Bennett M.J., Cakir M., McGregor M.J., Li Q., Meyer B., Roesch F., Vallet T., Kain A.M., Miorin L., Moreno E., Naing Z.Z., Zhou Y., Peng S., Shi Y., Zhang Z., Shen W., Kirby I.T., Melnyk J.E., Chorba J.S., Lou K., Dai S.A., Barrio-Hernandez I., Memon D., Hernandez-Armenta C., Lyu J., Mathy C.J., Perica T., Pilla K.B., Ganesan S.J., Saltzberg D.J., Rakesh R., Liu X., Rosenthal S.B., Calviello L., Venkataramanan S., Liboy-Lugo J., Lin Y., Huang X.P., Liu Y., Wankowicz S.A., Bohn M., Safari M., Ugur F.S., Koh C., Savar N.S., Tran Q.D., Shengjuler D., Fletcher S.J., O’Neal M.C., Cai Y., C.J.Chang J., Broadhurst D.J., Klippsten S., Sharp P.P., Wenzell N.A., Kuzuoglu D., Wang H.Y., Trenker R., Young J.M., Cavero D.A., Hiatt J., Roth T.L., Rathore U., Subramanian A., Noack J., Hubert M., Stroud R.M., Frankel A.D., Rosenberg O.S., Verba K.A., Agard D.A., Ott M., Emerman M., Jura N., von Zastrow M., Verdin E., Ashworth A., Schwartz O., d’Enfert C., Mukherjee S., Jacobson M., Malik H.S., Fujimori D.G., Ideker T., Craik C.S., Floor S.N., Fraser J.S., Gross J.D., Sali A., Roth B.L., Ruggero D., Taunton J., Kortemme T., Beltrao P., Vignuzzi M., García-Sastre A., Shokat K.M., Shoichet B.K, & Krogan N.J. (2020). A SARS-CoV-2 Protein Interaction Map Reveals Targets for Drug-Repurposing. Nature, 583(7816), 459-468.
Publication 2020
Biological Assay Cell Lines Cell Nucleus Cells Clone Cells Cytotoxin DAPI Fluorescence Formaldehyde Freezing Infection inhibitors Kidney Lung Mycoplasma prisma SARS-CoV-2 Serum Sulfoxide, Dimethyl Technique, Dilution Tissue Culture Techniques Titrimetry Vero Cells Violet, Gentian Viral Proteins Virus Replication
5
Isolation and Characterization of Fibroblast-Like Synoviocytes
Cited 3 times

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Publication 2019
Antibodies Blood Vessel Cell Culture Techniques Cells Character Culture Media Fibroblasts Fibrosis Flow Cytometry Fluorescein-5-isothiocyanate Immunoglobulin Isotypes Light Microscopy Macrophage Monoclonal Antibodies Phosphates Synovial Membrane Thy-1 Antigens Tissue Culture Techniques Tissues

Most recents protocols related to «Tissue Culture Techniques»

1
Radiation Exposure on EFT-400 Skin Equivalents
EpiDerm Full Thickness 400 (EFT-400) skin equivalent tissue models from MatTek Cooperation [51 ] (Lot. 021918GSB) were used for all radiation exposure experiments. The EFT-400 system consists of normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), however without vascular structures. They form a multi-layered, differentiated specimen consisting of basal, spinous, granular and cornified cell layers, partly resembling the normal human epidermal as well as a dermal layer arrangement. The missing of microvascular structures represents a limitation of this in-vitro model. However, the model is well-established for testing to radiation toxicity and alterations. Furthermore, applying skin models for radiotherapy experiments is also an option for avoiding/reducing the use of animal models [52 (link)].
The medium (Dulbecco’s Modified Eagle’s Medium (DMEM)) containing epidermal growth factor, insulin, hydrocortisone and other proprietary stimulators of epidermal differentiation, gentamicin 5 μg/ml, amphotericin B 0.25 μg/ml, phenol red and lipid precursors) and the 6-well plates used were supplied with the EFT-400 kit, see also Fig 1(A). After delivery of the tissues, new 6-well plates were prepared by adding 2.5 ml of the medium to each well using sterile technique in a tissue culture bonnet. Using sterile forceps, the gauze cover was removed from the insert again and each EFT-400 tissue was inserted into a well. No agarose adhered to the insert. The medium was completely in contact with the underside of the membrane of the tissue insert.
The in-vitro skin models were cultured in a humidified incubator at 37°C and 5% CO2 and transferred usually every 24 hours. The medium was stored in a refrigerator at temperatures between 2 and 8°C and pre-warmed in a water bath before use.
Immuno-histochemical analyses were performed according to the standard protocols to detect characteristic changes that are typical in natural skin when exposed to radiation and occur regularly in the investigated model. However, for the in-vitro model some specific proliferation and ageing patterns [53 (link)] have to be considered.
Bromberger L., Heise B., Felbermayer K., Leiss-Holzinger E., Ilicic K., Schmid T.E., Bergmayr A., Etzelstorfer T, & Geinitz H. (2023). Radiation-induced alterations in multi-layered, in-vitro skin models detected by optical coherence tomography and histological methods. PLOS ONE, 18(3), e0281662.
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Publication 2023
Amphotericin B Animal Model Bath Blood Vessel Cells Eagle Epidermal growth factor Epidermis Fibroblasts Forceps Gentamicin Homo sapiens Hydrocortisone Insulin Keratinocyte Lipids Obstetric Delivery Radiation Exposure Radiation Sickness Radiotherapy Sepharose Skin Sterility, Reproductive Tissue, Membrane Tissue Culture Techniques Tissues Vertebral Column
2
MERS-CoV Titration and Sequencing
rMERS-CoV titers were determined by plaque assay on Huh7 (JCRB, No. JCRB0403) or MRC5 cells (CCL-171, ATCC). Huh7 cells were fixed after an incubation period of 3 days, whereas MRC5 cells were fixed after 4 days. The tissue culture infective dose 50 (TCID50) endpoint dilution method in Huh7 cells and the TCID50 was calculated by the Spearman–Kärber algorithm. All work with live MERS-CoV was performed in the biosafety level 3 facility at the Leiden University Medical Center (LUMC). In order to confirm the presence of the intended substitutions in the rMERS-CoV PLpro- and rMERS-CoVMA PLpro- coding sequences, RNA was isolated from virus-containing supernatants or lung homogenates with the QIAamp Viral RNA Mini Kit (Qiagen). Total RNA was reverse transcribed to cDNA using RevertAid H minus reverse transcriptase (Thermo Fisher Scientific) and random hexamers. The PLpro domain (nucleotides 4435-5409 of the MERS-CoV genome was amplified by PCR using Accuzyme DNA polymerase (Bioline) and after purification the PCR product was sequenced.
Myeni S.K., Bredenbeek P.J., Knaap R.C., Dalebout T.J., Morales S.T., Sidorov I.A., Linger M.E., Oreshkova N., van Zanen-Gerhardt S., Zander S.A., Enjuanes L., Sola I., Snijder E.J, & Kikkert M. (2023). Engineering potent live attenuated coronavirus vaccines by targeted inactivation of the immune evasive viral deubiquitinase. Nature Communications, 14, 1141.
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Publication 2023
Biological Assay Cells Dental Plaque DNA, Complementary DNA-Directed DNA Polymerase Exons Genome Lung Middle East Respiratory Syndrome Coronavirus Nucleotides RNA, Viral RNA-Directed DNA Polymerase Technique, Dilution Tissue Culture Techniques Tissues Virus
3
SARS-CoV-2 and Influenza A Virus Quantification
For SARS-CoV-2, viral RNA was extracted 24 h post-infection from the supernatants of infected cells and copy numbers of the viral gene E were determined by TaqMan OneStep RT-qPCR with E_Sarbeco primers and probe [19 (link)] and following instructions of the QIAGEN QuantiNova Probe RT-PCR Kit. At 24 h post-infection, infectious particles were also titrated from the supernatants of infected cells by the tissue culture infectious dose 50% (TCID50) method on Vero E6 cells. For influenza A virus, infectious virus was titrated from the supernatants of infected cells 24 h post-infection by the TCID50 method on MDCK cells.
Foret-Lucas C., Figueroa T., Bertin A., Bessière P., Lucas A., Bergonnier D., Wasniewski M., Servat A., Tessier A., Lezoualc’h F, & Volmer R. (2023). EPAC1 Pharmacological Inhibition with AM-001 Prevents SARS-CoV-2 and Influenza A Virus Replication in Cells. Viruses, 15(2), 319.
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Publication 2023
Cells Infection Influenza A virus Madin Darby Canine Kidney Cells Oligonucleotide Primers Reverse Transcriptase Polymerase Chain Reaction RNA, Viral SARS-CoV-2 Tissue Culture Techniques Vero Cells Virus
4
Biofilm Detection in S. aureus
The tissue culture plate method was employed for detection of biofilm-producing S. aureus and a quantitative test was performed as published elsewhere [14 , 18 ].
Saud B., Khatri G., Amatya N., Paudel G, & Shrestha V. (2023). Methicillin-Resistant and Biofilm-Producing Staphylococcus aureus in Nasal Carriage among Health Care Workers and Medical Students. The Canadian Journal of Infectious Diseases & Medical Microbiology = Journal Canadien des Maladies Infectieuses et de la Microbiologie Médicale, 2023, 8424486.
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Publication 2023
Biofilms Tissue Culture Techniques
5
Validating Optimized Callus Formation
The reliability of the predicted-optimized results obtained through the genetic algorithm was evaluated through a validation experiment conducted in the laboratory. The validation experiment aimed to confirm the accuracy and reproducibility of the optimized combinations of phytohormones in achieving high callus formation rates and callus fresh weights. A total of seven replications were performed to ensure robustness and statistical significance. For each replication, the optimized phytohormone combinations determined by the genetic algorithm were used as treatment groups. Specifically, the combination of 1.31 mg/L BAP, 1.02 mg/L KIN, 1.44 mg/L NAA, and 1.70 mg/L IBA was tested for callus formation rate, while the combination of 1.39 mg/L BAP, 0.81 mg/L KIN, 1.22 mg/L NAA, and 1.60 mg/L IBA was evaluated for callus fresh weight (Fig 1C and 1D). The plant material and the tissue culture procedures, including explant preparation, media preparation, and incubation conditions, followed standard protocols as described for the first trial experiment. The data collected from the validation experiment were analyzed statistically to compare the results with the predicted-optimized outcomes of the genetic algorithm.
Rezaei H., Mirzaie-asl A., Abdollahi M.R, & Tohidfar M. (2023). Enhancing petunia tissue culture efficiency with machine learning: A pathway to improved callogenesis. PLOS ONE, 18(11), e0293754.
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Publication 2023
1-naphthylarsonic acid Callosities DNA Replication MAGI1 protein, human Plant Growth Regulators Plants Tissue Culture Techniques

Top products related to «Tissue Culture Techniques»

1
Fetal bovine serum (fbs) by Thermo Fisher Scientific
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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.
2
Dulbecco modified eagle medium (dmem) by Thermo Fisher Scientific
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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.
3
Penicillin streptomycin by Thermo Fisher Scientific
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
4
Penicillin by Thermo Fisher Scientific
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Penicillin is a type of antibiotic used in laboratory settings. It is a broad-spectrum antimicrobial agent effective against a variety of bacteria. Penicillin functions by disrupting the bacterial cell wall, leading to cell death.
5
Streptomycin by Thermo Fisher Scientific
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Streptomycin is a broad-spectrum antibiotic used in laboratory settings. It functions as a protein synthesis inhibitor, targeting the 30S subunit of bacterial ribosomes, which plays a crucial role in the translation of genetic information into proteins. Streptomycin is commonly used in microbiological research and applications that require selective inhibition of bacterial growth.
6
Fetal bovine serum (fbs) by Merck Group
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FBS, or Fetal Bovine Serum, is a commonly used cell culture supplement. It is derived from the blood of bovine fetuses and provides essential growth factors, hormones, and other nutrients to support the growth and proliferation of a wide range of cell types in vitro.
7
Penicillin by Merck Group
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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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Streptomycin is a laboratory product manufactured by Merck Group. It is an antibiotic used in research applications.
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Dulbecco modified eagle medium (dmem) by Merck Group
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DMEM (Dulbecco's Modified Eagle's Medium) is a commonly used cell culture medium formulated to support the growth and maintenance of various cell lines. It provides a balanced salt solution and essential nutrients required for cell proliferation. DMEM is suitable for use in a wide range of cell culture applications.
10
Collagenase type 1 by Merck Group
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Collagenase type I is an enzyme used in laboratory settings to break down collagen, a structural protein found in various tissues. It is commonly used in cell isolation and tissue dissociation procedures.

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