Cell culture technology notes big summary

Before starting the experiment, the sterile room and laminar flow hood should 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. Only one cell line should be handled per session, and even if the same medium is used, it should never be shared between different lines to prevent cross-contamination. After completing the experiment, all materials should be removed from the workbench, and the area should be disinfected again with 70% ethanol. There should be a minimum of 10 minutes between operations to maintain sterility. The aseptic working area must remain clean and uncluttered. Essential items like test tube racks, pipette holders, or straw boxes can be placed temporarily, but other lab supplies should be removed after use to avoid blocking airflow. All materials brought into the sterile workstation should first be wiped with 70% ethanol. Experimental procedures should be conducted in the central sterile zone of the workbench, avoiding the non-sterile edges. This helps reduce the risk of contamination. When handling sterile materials, care must be taken to avoid touching the tips of pipettes or the mouth of containers. Never perform experiments directly over open containers. Once a container is opened, hold the cap with one hand and the bottle body with the other, and keep the bottle at a 45-degree angle when pouring. Avoid placing the cap on the bench with the opening facing up to prevent contamination. All personnel must wear lab coats and gloves during experiments. Special attention should be given when working with human-derived or virus-infected cell lines, and a Class II biosafety cabinet should always be used. Be cautious of aerosol generation, toxic substances such as DMSO or TPA, and sharp objects like needles to ensure personal safety. Regular maintenance checks are essential. These include monitoring CO2 cylinder pressure, checking the CO2 incubator for correct temperature, CO2 concentration, and ensuring that the water tray is filled with sterile water and replaced weekly. The airflow pressure in the laminar flow hood should also be checked regularly, along with the replacement schedule for UV lamps and HEPA filters (pre-filter every 300 hours, HEPA filter every 3000 hours). Disinfectant (Zephrin at 1:750 dilution) can be added to the sink, and the water should be changed frequently to maintain cleanliness. Regarding trypsin storage: It is typically stored at -20°C in 50 mL aliquots. When ready to use, a single aliquot can be thawed at 4°C. If stored at 4°C, trypsin should not be kept for more than a few days, as prolonged exposure may reduce its activity. It’s best to use it fresh whenever possible. For media prepared with serum, it is generally recommended to store them at 4°C for no more than one month. While storage at -20°C might extend the shelf life, it’s better not to exceed 3–4 months. Primary cells are more sensitive, so shorter storage times are preferable to maintain viability. Cleaning glassware involves soaking in tap water for 30 minutes, followed by washing with detergent and ultrasonic cleaning for about 30 minutes. After rinsing thoroughly, soak in chromic acid solution for 6–18 hours, rinse with tap water 10–15 times, and finally wash with distilled water 3–4 times before autoclaving. Most plastic items, such as culture caps, pipette tips, and rubber stoppers, can be autoclaved. Reusable items like imported plates or dishes can be cleaned and UV-sterilized for 1–1.5 hours before reuse. However, plastic culture bottles are difficult to clean and should ideally not be reused. If necessary, they can be treated with ethylene oxide, but must be left undisturbed for at least six months afterward. Under a light microscope, epithelial cells often appear in a "paving stone" pattern, with elongated processes connecting distant cells. They exhibit growth characteristics. In contrast, interstitial cells are usually spindle-shaped and do not grow actively. Their morphology can change depending on culture conditions—HeLa cells, for example, may become fusiform under acidic conditions.

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