Cell School | Culture K7m2-WT cells, don’t miss this knowledge

2022-06-05 0 By

K7m2-wt (mouse osteoblasts for osteosarcoma) formed tumors in Balb/ C mice and spontaneously metastasized to the lungs in over 90% of inoculated mice.K7m2-wt cell antigen expression: ⅷ factor, integrated sialic acid protein (BSP), diglycan, gelatinoglycan, villin.In addition, the expression of bone sialic acid protein, diglycan, agglutinoglycan and osteomodulin showed the characteristics of bone family cells in K7m2-WT cells.Basic information Cell recovery 01 Preheat the water bath to 37℃, prepare clean disposable gloves, take the cells out of the liquid nitrogen tank, put them into disposable gloves, quickly put them into the water bath, shake the frozen storage tube to accelerate dissolution, it is appropriate to dissolve all within one minute;02 Place thawed cells in a centrifuge tube with fresh culture medium and wipe the outside of the tube with 75% alcohol before opening the lid;03 Centrifuge at 1200rpm for about 3 minutes, and remove the supernatant after centrifugation.04 Take an appropriate amount of complete culture medium matching the cells to suspend the cells, connect the cells into a sterile container, replenish a certain amount of culture medium, and then put the cells into an incubator for culture.Cell cryopreservation 01 Configures cryopreservation fluid.Because DMSO will be hot when configured, it is necessary to wait for the frozen storage liquid to be cold before using, to avoid burning cells;After digested, the cells were terminated with fresh culture medium, and cell suspension was prepared, centrifuged at 1200rpm for about 3 minutes, and the supernatant was absorbed as far as possible.03 Add the configured cryopreservation solution, adjust the concentration to about 1×106 cells /mL, and divide into cell cryopreservation tubes;04 The freeze-stored liquid shall be transferred to the program freeze-storage box and placed in the -80℃ refrigerator overnight.Take out the cryopreservation tube from the -80℃ refrigerator and quickly transfer it to liquid nitrogen for long-term preservation.Cell passage 1: The medium was sucked out and discarded, and 2mL PBS was added for rinsing;2. Suck out PBS, discard, add 2mL trypsin and rinse;3. Put them into the incubator for digestion for 2-3 minutes. Gently tap the side wall of the petri dish to see that the cells are off the wall and the aggregated cell clusters are loose and open, then add 2mL complete medium to terminate digestion;4 Blow the cells gently, make the cells mixed into suspension, inoculate according to the demand.Tips Cell density should not be too low;Make sure that all cells are infiltrated when rinsing.Normal growth of K7m2-WT cells Precautions for normal growth and culture of K7m2-WT cells 1 The cells are sensitive to external stimuli, so it is recommended to use fresh DMEM high sugar medium. When the medium turns purple, it is not recommended to continue using the medium, and replace the fresh medium in time;2 The cell is not firmly attached to the wall, and some cells will be suspended.When changing the liquid, do not rinse with PBS. When too many suspended cells are collected by centrifugation and then put back to the original culture bottle;3 Please pay attention to the degree of digestion during the passage of this cell, do not overdigest.