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MARIETTA BIOL 309 - B16 cell culture

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Cell Culture and Fluorescent Staining Page 1 Culturing and Fluorescent Staining of B16 Melanoma Cells In this lab exercise you will learn how animal cells can be grown in culture and used to analyze the cytosolic arrangement of actin filaments. Cell culture is a widely used methodology for studying the behavior of cells independent of the variables existing within the whole organisms. The technique allows investigation of intracellular processes for both normal and abnormal cells (such as cancerous cells), while allowing analysis of cellular responses to experimental modifications. Animal cell culture is one of the most important approaches to the study cell structure and is widely used in applied applications such as in the production of vaccines, antibodies and other commercially important biological products. A primary culture is created when the cells placed into culture are taken directly from an animal source, typically by mechanically disrupting a tissue source and allowing the cells to disperse onto the culture medium. Although sometimes a suspension culture may be produced, in which the cells remain free-floating in the medium, more commonly the culture will eventually form a monolayer of cells attached to lower surface of the culture flask. Animal cells are typically cultured using specially designed culture flasks in a solution containing a complex mixture of nutrients and supplemented with additional hormonal substances. For example B16 cells are cultured in CDMEM – which consists of DMEM, Dulbecco’s Modified Eagle’s Medium (see Table 1) to which is added FBS (fetal bovine serum), to provide essential growth hormones, and the antibiotics penicillin and streptomycin, to discourage bacterial contamination. Fortunately many types of cell culture media, such as DMEM, are commercially available as a premixed formulation. Every several days some of the cells must be caused to detach and subcultured (“passaged”) to fresh medium, yielding a secondary culture. Some animal cell lines, such as human HeLa cells and mouse B16 have been maintained in culture for many decades. Normal cells generally display contact inhibition when grown in culture, meaning that when the cells have become a confluent monolayer (and in direct contact with other cells) they stop dividing. Furthermore, normal cells (other than stem cells) will divide only for a limited number of times before ceasing to grow. Unlike normal cells, some cultured cells are said to be immortal, i.e., they will grow continuously in culture and often lack contact inhibition. A transformed state is typical of many types of cancerous cells, such as HeLa and B16. Objectives The objectives of this lab exercise are for you to learn: - basic principles of animal cell culture. - aseptic culture technique to maintain a continuous cell line over several weeks. - how fluorescent labeling can be used to study distribution of cytoskeletal filaments.Cell Culture and Fluorescent Staining Page 2 CDMEM composition DMEM (Sigma-Aldrich, D6429) 10% FBS (PAA labs, A15-507) 1% Penicillin/Streptomycin (Sigma-Aldrich, P4333) PBS (Sigma-Aldrich, D8537) 2.7 mM KCl 8.1 mM Na2HPO4 1.5 mM KH2PO4 137 mM NaCl Trypsin/EDTA (Sigma-Aldrich, T4174) 0.1% Trypsin 0.4% EDTA•4Na 0.9% NaClHow are animal cells cultured? The culture conditions we will use are typical for many different types of cultured cells. The cells are grown in plates containing CDMEM medium. The cultures are maintained in an incubator at 37OC supplemented with 5% CO2 in the atmosphere and containing a tray of water to maintain humidity. “Passaging” refers to the transfer of cells from one culture plate to another. This is performed by causing the cells to detach from the culture plate and then transferring a sample to a new culture well with fresh medium. The wells to be used for each round of subculturing are shown in Figure 1. The cells are caused to detach with a solution of trypsin-EDTA. Trypsin is a protease that gently cleaves cell surface proteins adhere the cells to the plate surface; EDTA is a chelating compound that binds to calcium, which also promotes cell release. One of the challenges to performing cell culture is the need to prevent bacterial or fungal contamination of the culture medium, and you will need to take many precautions when the cells are being passaged. We will culture B16 cells in 6 well culture plates. Supplies needed include: CDMEM – screw-capped bottles B16-F10 cells (ATCC #CRL-6475) 12 well culture plate (Corning #3336) Trypsin/EDTA– in 13 mm brown cap tube -- thaw before using PBS – small bottle P-1000 pipetter & sterile tips sterile Petri plate 100 ml beaker – for solution waste discard Inverted microscopes General procedures for maintaining sterile cultures Before performing any procedure with cell culture the workspace must be carefully cleaned. 1. Wipe down your workspace with disinfectant. 2. Wipe shaft of micropipetter with disinfectant. 3. Have all supplies available on your bench before beginning. 4. Wash your hands and arms. To avoid contamination when working with cultured cells, good aseptic technique must be applied, which includes: 1. Do not open your culture plate more than necessary to add or remove samples. 2. Immediately recover the culture plate between procedural steps. 3. Immediately dispose of a pipet or pipet tip that touches an unintended surface. 4. Dispose of pipets or pipet tips between each operation. 5. Store lids of tubes and vials face down in the sterile Petri plate. 6. After removing the cap of a tube or vial, always hold it at an angle. 7. Never touch the lip of a tube or vial.Cell Culture and Fluorescent Staining Page 3 Figure 1. Passaging cells to new culture wells.Procedures for passaging the cells Passaging of cells is not to be performed during regular lab periods. Cells should be transferred on an as-needed basis (approximately every 3 days) by monitoring cell density with the inverted microscope. Cells should be transferred when they reach approximately “80% confluence”. *** Keep a full record all of your culturing activities in your lab notebook. *** 1. Trypsinizing to release cells from culture well 1. Prep the work space as described above; be sure to wash your hands and arms. Remove CDMEM from refrigerator, and thaw Trypsin/EDTA. 2. Remove the media from the well with the cells. 3. Add 1 ml of PBS, and


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