Cell culture technology notes big summary

Before starting the experiment, the sterile room and laminar flow hood must be sterilized using a UV lamp for 30 to 60 minutes. The work area should then be wiped with 70% ethanol, and the laminar flow fan should be turned on for at least 10 minutes to ensure proper air circulation before beginning any procedure. Each operation should handle only one cell line at a time, and even if the same medium is used, it should never be shared between different cell lines to prevent cross-contamination. After completing the experiment, all test materials should be removed from the workbench, and the area should again be disinfected with 70% ethanol. It is important to maintain an interval of more than 10 minutes between operations to allow the aseptic environment to remain stable. The aseptic work area should always be kept clean and uncluttered. Essential items such as test tube racks, pipette tips, or suction devices may be temporarily placed on the bench, but all other materials should be removed after use to ensure good airflow. Before bringing any item into the sterile workstation, it should be wiped with 70% ethanol. All experimental procedures should be conducted in the central sterile zone of the workbench, avoiding the edges where contamination risk is higher. When handling sterile materials, care must be taken to avoid any contact with the tip of pipettes or the mouth of containers. Never operate directly over open containers, and when opening a bottle, hold the cap with your hand while keeping the bottle body steady at a 45-degree angle. Avoid placing the cap on the table with the opening facing up to reduce the chance of contamination. Lab personnel must wear lab coats and gloves during all experiments. Special attention should be given when working with human-derived or virus-infected cell lines, requiring at least a Class II biosafety cabinet. During the process, avoid creating aerosols, handle toxic substances like DMSO and TPA with caution, and take care to prevent injuries from sharp objects such as needles. Regular maintenance checks are essential: check CO2 cylinder pressure, monitor CO2 incubator settings (including CO2 concentration, temperature, and water level in the tray—always use sterile water and replace weekly), and ensure the airflow pressure in the biosafety cabinet is adequate. Replace UV lamps and HEPA filters according to manufacturer guidelines (pre-filter every 300 hours, HEPA filter every 3000 hours). Disinfectant (Zephrin 1:750) can be added to the sink, and the water should be changed regularly to maintain cleanliness. Regarding trypsin storage: it is typically stored at -20°C in small aliquots (e.g., 50 mL screw tubes). When ready to use, take one tube and place it in a 4°C refrigerator. If stored at 4°C, trypsin can last for several weeks, but its activity may decrease over time if left too long. It's best to use freshly thawed trypsin whenever possible. For reagents like powdered media, once prepared with serum, they should not be stored for more than one month at 4°C. Although storing at -20°C might extend the shelf life, it's generally better not to exceed 3-4 months. For primary cell cultures, which are more sensitive, shorter storage times are recommended to maintain viability and performance. Cleaning and disinfecting lab equipment is crucial. Used glassware should first be soaked in tap water for 30 minutes, then cleaned with a mild detergent and ultrasonically washed for about 30 minutes. If no ultrasonic cleaner is available, a soft brush can be used. After rinsing thoroughly, soak the glassware in chromic acid solution for 6–18 hours, rinse with tap water 10–15 times, and finally wash with double-distilled water 3–4 times. Dry and autoclave before reuse. Many plastic items, such as culture caps, rubber stoppers, and pipette tips, can be autoclaved. However, some disposable plastics are designed for single use. If reusing expensive items like imported plates or dishes, they can be cleaned and sterilized under UV light for 1–1.5 hours. Plastic culture bottles are difficult to clean and should ideally not be reused. If necessary, ethylene oxide sterilization is an option, but these items must be left to ventilate for at least six months after treatment. Under a light microscope, epithelial cells often appear in a "paving stone" arrangement, with elongated processes connecting distant cells. These cells exhibit growth characteristics. In contrast, interstitial cells tend to be spindle-shaped and do not show active growth. However, cell morphology can change depending on environmental conditions. For example, HeLa cells, which are epithelial, may adopt a fusiform shape under acidic culture conditions.

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