Let’s talk about how to keep your cell lines free from contamination. I’m Geena, research associate at Cell Signaling Technology, and this is CST Tech Tips. If you use cell lines or primary cells in your experiments, you know that sterile technique is crucial to prevent bacteria, fungi, and mycoplasma from contaminating your cells, and ruining your experiment. Today, I’ll review the basic dos and don'ts of sterile technique, also known as aseptic technique. Good sterile technique will help your experiments to run smoothly and generate reproducible data. Because microorganisms are in the air and on surfaces in the lab, cell culture tasks must be done in a sterile environment to prevent contamination. The biosafety cabinet, also known as cell culture hood or tissue culture hood, uses laminar air flow as a barrier to reduce contamination by airborne particles in the room. Plastic consumables, liquid media, and other reagents for cell culture are available in sterile packaging. The sterility of these components is important, but the most crucial factor is your technique. Before starting, wash your hands and put on PPE including lab coat, long sleeves, gloves, and safety glasses. These reduce risk of contamination and offer protection. Always adhere to biosafety guidance from your local lab safety officer depending on the materials you will be working with, including disposal of waste. Turn on the cabinet lights and air filter, adjust the glass sash height to the specified mark, and run the filter for at least 15 minutes before starting any work. Use a spray bottle to apply 70% ethanol to your gloved hands. Then, spray the interior of the biosafety cabinet and wipe with lint free wipes or paper towels. To minimize disruption of the air curtain, avoid moving your arms laterally when possible. Instead enter and exit the front of the cabinet straight on. Every time you place an item in the cabinet, you must also disinfect it by spraying with ethanol and wiping. This includes packaged consumables, media and reagent bottles, paying special attention to the cap and neck, and smaller conical and micro tubes. If you use a water bath to pre-warm bottles, use bactericidal and fungicidal additives in the water and replace the water regularly. Don’t clutter the cabinet with too many items. Overcrowding can disturb the air curtain barrier. Plan ahead, and use only what you need for each step. You can keep the pipettor and consumables to one side, depending on if you are right- or left-handed. Other items can be placed towards the back of the cabinet, leaving the center as your working area. When removing a bottle or tube cap, don’t place it face up. Place it on the surface face down. You may use your non-dominant hand to hold the cap while pipetting, but this takes practice and dexterity. To use serological pipettes, open the end of the wrapper, place the end in the pipettor firmly, and rotate so you can see the gradations. Avoid contacting the pipette tip to the exterior of any vessels, and avoid contact with the interior surfaces of the bottle or flask, especially the neck or cap. If you inadvertently get drops of liquid on the outside of a vessel or in the cabinet, spray a wipe with ethanol, and clean up the drops. To reduce risk of cross contamination, use each tip only once, and then dispose. Don’t re-use it. Replace caps onto flasks, bottles, or tubes as quickly as possible, and take care they are threaded and closed properly. Label your cell culture vessels and any bottles or tubes as needed before removing them from the hood. Try to move through each step deliberately and efficiently, but at the same time, allow yourself enough time and don’t rush, as this only makes human error more likely. As with any skill, experience and practice are key to becoming proficient. After removing all your items, wipe the interior of the cabinet once more with ethanol. The cell culture room is usually a shared resource, so it’s always a good idea to be considerate of your coworkers when scheduling time in the biosafety cabinet. Keep tidy, and communicate about any issues that may arise so that everyone can keep their experiments running. Thanks for watching! If you found this helpful, please hit the like button and subscribe to our channel for more Tech Tips. You can also find content to guide your scientific career at cellmentor.com, a resource from CST and Cell Press, and sign up for their newsletter. If you have any questions about a CST antibody, contact a scientist at cellsignal.com/support. We’ll see you next time, and good luck with your experiments!