Basic Techniques in Mammalian Cell Tissue Culture

“CLIP analysis is a complex topic beyond the scope of this protocol but the omniCLIP peak caller (Drewe-Boss, Wessels, & Ohler, 2018) has been used for easyCLIP-style library preparation in a recent publication (Poetz et al., 2022).”

“Lift cells from T-25 flask using 0.5 ml trypsin (see Phelan & May, 2015) and plate on a poly-d-lysine-coated glass-bottom dish.”

“The primary cells were cultured using the tissue attachment method [19].”

“All the samples were dissolved in DMSO (Dimethyl sulfoxide) in a ratio of weight of sample to volume of solvent as follows: each 0.1 g of sample should be dissolved in 10 ml DMSO (15) .”

“…chain reaction (PCR; Kramer and Coen, 2000), agarose gel electrophoresis (Voytas, 2000), electroporation (Potter and Heller, 2010), qPCR (Bookout et al., 2006), and enzymatic dissociation of cells with trypsin/EDTA (Phelan and May, 2015) I. scCRISPR sgRNA and insertion homology-arm design 1.”

“…Sterile reagent reservoirs (VWR, cat. no. 89094-662) E-Plate Cardio 96 (ACEA Biosciences) Additional reagents and equipment for determining cell number (Phelan, 2007) Thaw, plate, and maintain iCell cardiomyocytes on 6-well plates The following steps must be performed in a sterile biosafety…”

“Freezing cells is the most effective way of bringing cells in a non-active state and allows for preserving cells for extended periods of time (months at 80 1C, years at 196 1C) (Phelan, 2007).”

“…2012), immunoblotting (western blotting; Ni et al., 2016), cell culture techniques including trypsinization and cell counting (see Current Protocols article: Phelan & May, 2015), and euthanasia of mice (see Current Protocols article: Donovan & Brown, 2006) Applying CAGE-prox to lethal factor 1.”

“Add 1 ml 0.05% trypsin-EDTA to each flask and incubate at 37°C for 5 to 10 min, allowing cells to detach from the surface of the flask (also see Current Protocols article: Phelan & May, 2015).”

“Complete blood cell counts can be obtained with the EDTA tube using an automated blood cell counter, or hemacytometer (see Phelan & May, 2016).”

“…and buffered with HEPES, if necessary 2-well Lab-Tek chambers (Nunc) Additional reagents and equipment for growing mammalian cells in culture (Phelan, 2007), preparing cell culture medium (Sato and Kan, 1998), and preparing and transfecting plasmids (see APPENDIX 3A) Assessment of Cell…”

“The results were analyzed at 570 nm absorbance using an ELISA microplate reader at 570 nm to measure cell samples (Uðuz and Nazýroðlu, 2012; Phelan and May, 2015).”

“The cells were counted using a hemocytometer after being harvested from sterile culture flasks, and trypan blue exclusion determined the viability of the cells (Phelan, 1998).”

“Tenocytes were stripped from surrounding tissues in the way described in [158].”

“25% Trypsin-EDTA solution [17] followed by addition of complete cell culture medium containing”

“Cell suspensions fixed with 4% PFA were frozen by three different methods: (1) by using 10% dimethylsulfoxide (DMSO) in complete RPMI media (10% FBS, 2mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate; Life Technologies, Grand Island, NY, USA) [10], (2) gradual dehydration using ethanol (50%, 70%…”

“References for general and best practice for cell culture can be found in several comprehensive reviews [14, 15, 16].”

“Normally cells are cultured in petri plates or flasks [1].”

“…laboratories working with cell cultures (Carrillo-Ávila et al. 2023a; Drexler & Uphoff 2002; Nims & Price 2017; Segeritz & Vallier 2017), as contamination often goes unnoticed due to the absence of visible turbidity or cytopathic effect (Cobo et al. 2007; Cor-ral-Vázquez et al. 2017; Phelan 2007).”

“5%), which cleaves cell surface proteins [25, 26].”

“…Orbital shaker Odyssey CLx Imaging system (Li-COR) Microsoft Excel and GraphPad Prism software Additional reagents and equipment for basic cell culture techniques including trypsinization and determining live cell density by trypan blue exclusion (see Current Protocols article: Phelan & May, 2015)”

“J L a b Clin M e d 130: 530534. 42. Duivenvoorden WC, Hirte HW, Singh G.(1997). Use of tetracy-”

“Variables linked to the culture conditions of the cells (for example the culture medium) are interesting to include in the model (Phelan 2007; Balls et al. 2006).”

“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 (Phelan, 1998).”

“Elles sont cultivées à 37 °C sous une atmosphère chargée de 5 % de C02 selon les protocoles standards en vigueur (Phelan, 2007).”