Cell Culture Basics from Gibco®
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The entire Cell Culture Basics training course is available online at http://www.invitrogen.com/cellculturebasics. The handbook and videos are intended as an introductory cell culture training course, covering topics such as getting familiar with the requirements of a laboratory dedicated to cell culture experiments, laboratory safety, aseptic technique, and microbial contamination of cell cultures, as well as providing basic methods for passaging, freezing, and thawing cultured cells.
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established. In this video, we present the basic equipment used in cell culture and proper way to set-up a laboratory. In this video, we present the basic equipment used in cell culture and proper way to set-up a laboratory.
Welcome to cell culture! Successful cell culture depends on upon many factors, including the quality of the cells and reagents you use, your sterile technique, the amount of experience you have and the right laboratory equipment.
Proper set up is important. Have everything you need close by. The basic equipment you’ll find in a cell culture lab includes a cell culture hood, an incubator, a water bath, a centrifuge, a refrigerator/freezer, a hemacytometer, and a microscope. You’ll also need cell culture dishes, medium and other reagents, pipettes and waste containers. The hood provides a sterile environment to work with your cells without fear of contamination. Always open your cell culture dishes and reagent bottles in a sterile environment.
Outside the cell culture hood, keep a stack of disposable pipettes nearby. Glass pipettes are placed in the hood during use, and only opened inside the sterile field. Have the trash, biohazard bin, and sharps container close by. A cell counting machine such as the Countess gives you quick, accurate counts needed during culturing. Or, you can count cells manually with a hemacytometer. You may also wish to have the centrifuge located in the cell culture facility.
Incubators provide the appropriate environment for cell growth. Carbon dioxide and temperature settings depend on cell type and medium selection. Mammalian cells are cultured at 37 degrees Celsius in the presence of 5-7% carbon dioxide with high humidity. Inside the incubator, keep your cell culture flasks evenly spaced and avoid crowding to ensure that the air inside the incubator moves freely. This helps to maintain temperature, humidity, and proper gas exchange. Don’t make very tall stacks of plates. They may fall over and lower ones may not get the appropriate gas exchange.
Store all media and reagents according to the instructions on the label. Some components are known to degrade when left at room temperature or exposed to light for extended times. If the medium will be stored in a glass front refrigerator or a cold room, wrap the bottles in tin foil or store them inside a box. Some labs warm their reagents to 37 degrees Celsius before use. Warming reagents should take 10-20 minutes, depending upon the size of bottles you are using. Others choose to use reagents warmed to room temperature or directly from four degree storage.
If you use a water bath, make sure to keep bottles upright with necks out of the water. Specially designed bottle weights are available for use with the GIBCO bottle. Alternatively, metallic bath beads can be used instead of water. Bath beads are a cleaner, greener alternative to the water bath. While waiting for reagents to warm, prepare the materials you will need to use. When working under the hood, make sure the hood sash is at the proper height. Correct chair height and body position prevent shoulder stress, and also helps prevent contamination.
Never breathe or talk into the hood. Wipe the hood with 70% ethanol before and after use. Each item in the hood should be sprayed with 70% ethanol and wiped clean. Items within the hood are usually placed as follows:
1. PipetAid in front
2. Reagents in back right.
3. Tube rack in the rear middle.
4. Small container to hold liquid waste in the rear left.
5. Your cell plates in the center.
Never leave reagents in the water bath longer than necessary. Excessive warming can lead to L-glutamine degradation and loss of activity in trypsin solutions. Gibco Glutamax and TrypLE are more stable versions of L-glutamine and trypsin.