Basic Techniques in Mammalian Cell Tissue Culture

Phelan, Mary C.

doi:10.1002/0471143030.cb0101s36

Cited by 142Viral Infectious Diseases and Gene Expression in InsectsCellular Mechanics and InteractionsCell Image Analysis Techniques

Basic Techniques in Mammalian Cell Tissue Culture

Phelan, Mary C.·2007·Current Protocols in Cell Biology

DOI 10.1002/0471143030.cb0101s36

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Abstract

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.Curr. Protoc. Cell Biol. 36:1.1.1‐1.1.18. © 2007 by John Wiley & Sons, Inc.

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Each cell line was cultured in antibiotics-free medium for a week before mycoplasma detection.
“In order to detect the mycoplasma contamination after harvesting, each cell line was cultured in an antibiotics-free medium for a week (at least 4-5 days) without exchanging the medium (Phelan 2006(Phelan , 2007)).”
From Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran (2016) · cites this paper as Phelan 2007

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Fluorescence Activated Cell Sorting (FACS) in Genome‐Wide Genetic Screening of Membrane Trafficking
Menasché, B. L., Crisman, L., Gulbranson, D. R., Davis, E. M., Yu, H., & Shen, J. (2018) · Current Protocols in Cell Biology · doi:10.1002/cpcb.68 · Cited by 9 · see citing works
“…with 50-μm pore size (e.g., Sysmex CellTrics filters, #04-004-2327) Sterile flow cytometry tubes (e.g., USA Scientific, #1450-0810) Additional reagents and equipment for cell culture techniques, including culturing, trypsinization, and counting of viable cells using trypan blue (Phelan, 2007) 1.”
BackgroundMethodInfluential
Functional Stem Cell Integration Assessed by Organotypic Slice Cultures
Herlenius, E., Thonabulsombat, C., Forsberg, D., Jäderstad, J., Jäderstad, L. M., Björk, L., & Olivius, P. (2012) · Current Protocols in Stem Cell Biology · doi:10.1002/9780470151808.sc02d13s23 · Cited by 8 · see citing works
Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate
Kawase, T., Kogami, H., Nagata, M., Uematsu, K., Okuda, K., Burns, D. M., & Yoshie, H. (2011) · Cryobiology · doi:10.1016/j.cryobiol.2011.03.004 · Cited by 10 · see citing works
Mass spectrometry-based identification of protein kinase substrates utilizing engineered kinases and thiophosphate labeling
Chi, Y., & Clurman, B. (2010) · Current Protocols in Chemical Biology · doi:10.1002/9780470559277.ch100151 · Cited by 10
Mining the effect of the neonicotinoids imidacloprid and clothianidin on the chemical homeostasis and energy equilibrium of primary mouse neural stem/progenitor cells using metabolomics.
Fotopoulou, E., Lykogianni, M., Papadimitriou, E., Mavrikou, S., Machera, K., Kintzios, S., Thomaidou, D., & Aliferis, K. (2020) · Pesticide Biochemistry and Physiology · doi:10.1016/j.pestbp.2020.104617 · Cited by 9 · see citing works
XPO1 target occupancy measurements confirm the selinexor recommended phase 2 dose
Crochiere, M., Hannus, S., Hansen, K., Becker, F., Baloglu, E., Lee, M. S., Kauffman, M., Shacham, S., & Landesman, Y. (2017) · OncoTarget · doi:10.18632/oncotarget.22801 · Cited by 9 · see citing works
“The inhibitory rate of cell growth was calculated using the formula: % growth inhibition = (1 – OD extract treated)/OD negative control x 100) [30].”
Method
Label‐Free Dynamic Mass Redistribution and Bio‐Impedance Methods for Drug Discovery
Grundmann, M. (2017) · Current Protocols in Pharmacology · doi:10.1002/cpph.24 · Cited by 9 · see citing works
The Selective Inhibitor of Nuclear Export Compound, Selinexor, Inhibits NF-κB and Induces Anti-Non-Small Cell Lung Cancer Activity Regardless of p53 Status
Crochiere, M. (2016) · doi:10.16966/2381-3318.126 · Cited by 9
“The inhibitory rate of cell growth was calculated using the formula: % Growth inhibition=(1-OD extract treated)/OD negative control × 100 [23].”
Method
Isolation, Culture, and Characterization of Primary Salivary Gland Cells
Su, X.-Y., Pillai, S., Liu, Y.-N., & Tran, S. D. (2022) · Current Protocols · doi:10.1002/cpz1.479 · Cited by 8
“…(Miltenyi Biotec) 70-μm cell strainers (Fisher Scientific, cat. no. 22363548) Humidified, 37°C, 5% CO2 cell culture incubator 37°C water bath Additional reagents and equipment for cell culture techniques including counting cells (see Current Protocols article: Phelan & May, 2015) Tissue cleaning 1.”
Method
Optogenetic Tools for Manipulating Protein Subcellular Localization and Intracellular Signaling at Organelle Contact Sites
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“Protocols for trypsinization and other general cell culture techniques are found in Current Protocols article Phelan & May (2015).”
Method
Understanding cell culture dynamics: a tool for defining protocol parameters for improved processes and efficient manufacturing using human embryonic stem cells
Kusena, J. W. T., Shariatzadeh, M., Thomas, R., & Wilson, S. (2021) · Bioengineered · doi:10.1080/21655979.2021.1902696 · Cited by 8
EGF ligand fused to truncated Pseudomonas aeruginosa exotoxin A specifically targets and inhibits EGFR‑positive cancer cells.
Hashimi, S., Grant, B., Alqurashi, N., Alowaidi, F., & Wei, M. (2018) · Oncology Report · doi:10.3892/or.2018.6685 · Cited by 8
“Routine methods were used for the culturing of cell lines (12).”
Protein Inhibition by Microinjection and RNA-Mediated Interference in Tissue Culture Cells: Complementary Approaches to Study Protein Function
Stout, J. R., Rizk, R., & Walczak, C. (2009) · Methods in molecular biology · doi:10.1007/978-1-59745-202-1_7 · Cited by 8
“For more in-depth information on media, cells, and maintenance techniques, please refer to (9,10).”
Background
RiPCA: An Assay for the Detection of RNA‐Protein Interactions in Live Cells
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The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line
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Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinocyte Migration.
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Proteomes, Their Compositions and Their Sources
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Cytotoxic effect of Zataria multiflora on gastric cancer cell line (AGS) and normal fibroblast cells
Janitermi, M., Nemati, F., & Mohammadpour, G. (2015) · doi:10.17776/csj.62670 · Cited by 7
“Three thousand cells from log phase cultures were seeded in 100 μl of RPMI medium supplemented with 10% fetal bovine serum per well of 96-well flat-bottom culture plates [17].”
Method
Cytotoxic effect of Zataria multiflora on cervical cancer cell line (HeLa) and normal fibroblast cells
Janitermi, M., & Nemati, F. (2015) · doi:10.17776/csj.88915 · Cited by 7
“Three thousand cells from log phase cultures were seeded in 100 μl of RPMI medium supplemented with 10% fetal bovine serum per well of 96-well flat-bottom culture plates [36].”
Method
Cytotoxic effect of Zataria multiflora on breast cancer cell line (MCF-7) and normal fibroblast cells
Janitermi, M., & Nemati, F. (2015) · doi:10.17776/csj.56546 · Cited by 7
“Three thousand cells from log phase cultures were seeded in 100 μl of RPMI medium supplemented with 10% fetal bovine serum per well of 96-well flat-bottom culture plates [13].”
Method
Expression and purification of the receptor‐binding domain of SARS‐CoV‐2 spike protein in mammalian cells for immunological assays
Ábrahám, E., Bajusz, C., Marton, A., Borics, A., Mdluli, T., Pardi, N., & Lipinszki, Z. (2023) · FEBS Open Bio · doi:10.1002/2211-5463.13754 · Cited by 6
“80 ° C. Tips & Tricks [26] and the guidelines of the Expi293 Expression System manual (https://www.thermoﬁsher.com) should be followed.”
Method
Mg doped ZnO containing silk nanocomposite scaffolds for biofilm prevention during alveolar bone regeneration
Shahid, A., Moeen, F., Habib, S., Arshad, A., Zeeshan, R., Chaudhry, A. A., Khalid, H., Alhamoudi, F. H., Akhtar, H., & Khan, A. F. (2024) · Materials Chemistry and Physics · doi:10.1016/j.matchemphys.2024.129911 · Cited by 6 · see citing works
Deciphering variations in the endocytic uptake of a cell-penetrating peptide: the crucial role of cell culture protocols
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“This can be performed by addition of a low amount of the protease trypsin (0.05-0.5%), which cleaves cell surface proteins (Huang et al. 2010; Phelan 2007).”
BackgroundInfluential
Utilization of discarded surgical tissue from ultrasonic aspirators to establish patient-derived metastatic brain tumor cells: A Guide from the Operating Room to the Research Laboratory
Martirosian, V., Deshpande, K., Lin, M., Jarvis, C. A., Yuan, E., Chen, T. C., Zada, G., Giannotta, S., Attenello, F., Chow, F. E., et al. (2021) · Current Protocols · doi:10.1002/cpz1.140 · Cited by 5
“…Eppendorf) Rotating plate shaker 37°C, 5% CO2 cell culture incubator Collagen-coated (Support Protocol 2) and non-coated T-75 tissue culture flasks Additional reagents and equipment for basic cell culture techniques including trypsinization (see Current Protocols article: Phelan & May, 2015) 1.”
MethodInfluential
Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma
Lopes, B. R. P., Ribeiro, A. G., da Silva, T. F., Barbosa, L. V., Jesus, T. I., Matsuda, B. K., Costa, M. F., & Toledo, K. A. (2020) · Brazilian Journal of Biology · doi:10.1590/1519-6984.215721 · Cited by 5 · see citing works
Cellular Detection of G‐Quadruplexes by Optical Imaging Methods
Amor, S., Yang, S. Y. C., Wong, J. M. Y., & Monchaud, D. (2017) · Current Protocols in Cell Biology · doi:10.1002/cpcb.29 · Cited by 5 · see citing works
“…of a special type of four-way DNA junction named the Holliday junction; Liu & West, 2004), Gquadruplexes (G4) have attracted most of the research interest due to their involvement in key cellular processes such as replication, transcription, and translation (Maizels, 2015; Rhodes & Lipps, 2015).”
Background
Murine Retrovirally-transduced Bone Marrow Engraftment Models of MLL-fusion-driven Acute Myelogenous Leukemias (AML)
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“…a 4× objective 6-well cell culture dishes (Corning) 35-mm plates (Falcon) Additional reagents and equipment for cell culture including cell counting (Phelan & May, 2015), fluorescence activated cell sorting (FACS; Robinson et al., 2017), injection of mice (Donovan & Brown, 2006b), and euthanasia…”
Method
Combining Cytotoxicity Assessment and Xenopus laevis Phenotypic Abnormality Assay as a Predictor of Nanomaterial Safety
Al-Yousuf, K., Webster, C., Wheeler, G., Bombelli, F. B., & Sherwood, V. (2017) · Current Protocols in Toxicology · doi:10.1002/cptx.25 · Cited by 5
“Mammalian cell stocks for this assay are maintained and prepared using GLP as described above (Basic Protocol 2, step 1; see APPENDIX 3B, Phelan & May, 2016).”
Method
Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders.
Fernandez-Guerra, P., Lund, M., Corydon, T., Cornelius, N., Gregersen, N., Palmfeldt, J., & Bross, P. (2015) · JIMD Reports · doi:10.1007/8904_2015_494 · Cited by 5
“…analysis with *P value <0.05; **P value <0.01; ***P value <0.001 Cell viability is used to evaluate the amount of living cells in a cell population under treatments or to determine whether the expression of a mutation affects cell proliferation or promotes cell death (Phelan 1998; Bie et al. 2011).”
BackgroundMethodInfluential
Methods for Culturing Human Embryonic Stem Cells in a Xeno-Free System
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Deciphering the role of siRNA in anxiety and depression
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“This was especially relevant (and observed) compared with the solvent control and could be explained by a loss of cell mass due to starvation stress and cell detachment due to high medium dilution and a very low pH [32,35].”
Background

Authors: Phelan, M. C.
Affiliations: Molecular Pathology Laboratory Network (United States) (US)
Corresponding author: Phelan, Mary C.
Venue: Current Protocols in Cell Biology
Publisher: Wiley
Published: 2007-09
Volume: 36
Issue: 1
Pages: Unit 1.1-Unit 1.1
ISSN: 1934-2500 (ISSN-L)
1934-2616
DOI: 10.1002/0471143030.cb0101s36
OpenAlex: W2911622295
Cited by: 87
References: 0
Citations by year: 2026: 1 · 2025: 2 · 2024: 2 · 2023: 1 · 2022: 1 · 2021: 3 · 2020: 4 · 2019: 2 · 2018: 7 · 2017: 5 · 2016: 9 · 2015: 7 · 2014: 6 · 2013: 8 · 2012: 11
FWCI: 1.45
Type: journal article
Language: en
Open access: closed
Licence: onlinelibrary.wiley.com/termsAndConditions#vor (vor, 14 d)
Locations: Current Protocols in Cell Biology (publishedVersion)
PubMed (publishedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
Keywords: mammalian cell culture, aseptic technique, culture media preparation, subculture, viable cell count, cell preservation
MeSH terms: Animals, Cell Count, Cells, Cultured, Culture Media, Humans, Indicators and Reagents, Mammals, Suspensions, Transportation, Cryopreservation, Cell Culture Techniques

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Basic Techniques in Mammalian Cell Tissue Culture

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