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| 購買進口儀器、試劑和耗材——就在始于2001年的畢特博生物 www.mirosound.cn |
IntroductionNatural killer (NK) cells are crucial immune cells that can recognize and kill tumors and virus infection. It is difficult to obtain the large numbers of NK cells ex vivo that are necessary for adoptive immunotherapy. Several methods of NK activation/expansion have been reported that use expansion medium plus various cytokines (e.g., IL-2, IL-7, and IL-15). However, these require further optimization by researchers. The Corning NK kit contains a pre-coated T-75 flask, 50 mL NK Primary medium, 1.8 mL NK Primary supplement, and 1L NK Expansion medium. The first three components are specially designed for NK cell activation, whereas the NK Expansion medium is utilized for NK cell expansion. Both NK Primary medium and NK Expansion medium are manufactured using high quality reagents and cGMP-grade raw materials. The only proteins present in the media are injectable levels of serum albumin and recombinant human insulin.
For information on proliferation rates, immunophenotyping, and CCK-8 cytotoxicity testing using cells activated and expanding using this kit, see the Corning Application Note Human Natural Killer Cell Expansion Using Corning NK Expansion Kit (CLS-CG-AN-427). NK Cell Activation and Expansion?? Centrifuge the anti-coagulated blood at 400 xg for 10 minutes at room temperature (RT) and then transfer the plasma (on the top layer) into a new tube. ?? Heat inactivate the auto-plasma at 56°C for 30 minutes and then centrifuge at 800 xg for 20 minutes to remove the precipitate. The supernatant plasma should be stored at 4°C, which will be used up in the following 14 culture days. ?? Add equal volume of PBS (Phosphate Buffer Saline without calcium and magnesium, Corning Cat. No. 21-040-CV) as replacement of the removed auto-plasma to maintain a constant volume and resuspend the haemocytes gently. Note: Pre-adding 0.1% human serum albumin (HSA) in PBS helps to maintain haemocyte viability. ?? Prepare peripheral blood mononuclear cells (PBMCs) from the above blood sample using lymphocyte separation medium (LSM, Corning Cat. No. 25-072-Cl) according to manufacturer’s directions. Note: Use freshly collected human blood (within 2 hours of collection) for better performance; do not use blood that is drawn more than 24 hours prior to use. ?? Wash the PBMCs with at least 5-fold PBS without calcium and magnesium. Centrifuge at 500 xg for 10 minutes at RT to collect the PBMCs. ?? Dilute the PBMCs to 1 x 106 cells/mL using NK Primary medium containing 1.8 mL NK Primary supplement and 10% auto-plasma.
?? Add 30 mL of the PBMC suspension to the rinsed pre-coated T-75 flask and incubate at 37°C in a humidified atmosphere of 5% CO2 in air.
Note: Before Day 6, if the medium turns yellow with the cell density above 2.0 x 106 cells/mL, fresh NK Primary medium plus 10% auto-plasma should be added into the cell suspension to keep the cell density above 5 x 105 cells/mL.
?? On Day 6, centrifuge and resuspend the cells with an appropriate volume of NK Expansion medium containing 1,000 IU/mL IL-2 and 10% auto-plasma and then transfer to a new T-225 flask or gas- permeable culture bag.
?? At an interval of 2 or 3 days, based on the cell proliferation status, add fresh NK Expansion media containing 1,000 IU/mL IL-2 into the culture system and keep the cell density in the recommended density range from 5 x 105 to 2.0 x 106 cells/mL.
Note: In the NK expansion stage, 0.5% to 1% auto-plasma is recommended to be contained in the freshly added media until it has been exhausted.
?? Harvest NK cells around Day 14.
Note: Blow the cell suspension gently and tap the culture vessels softly to avoid cell damage throughout the entire experimental process to maintain cell viability.
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購買進口儀器、試劑和耗材——就在始于2001年的畢特博生物
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