U87/DK_Luc熒光示蹤穩定表達細胞株-BioVector NTCC保藏中心
I. Introduction Host Cell: U87/DK Expressed gene: Luciferase Stability: 32 passages Freeze Medium: 90% FBS+10% DMSO Culture Medium: DMEM+10%FBS+1ug/ml Puromycin Mycoplasma Status: Negative Storage: Liquid nitrogen II. Description of Host Cell Line Organism: Homo sapiens (Human) Tissue: Brain Disease: Glioblastoma Morphology: Grown as monolayer, morphology epithelial like Growth Properties: N/A III. Representative Data
Figure. Detect Luciferase assay by Promega Bright-Glo Luciferase Assay System. The number of Luminescence signal is 565±13(Mean±SD) on U87/DK cells while 640080±27416(Mean±SD) on U87/DK-Luc cells.
IV. Stablity Test
Figure.Stablity test results. U87/DK-Luc cells showed at least 32 passage stability with similar luminescence unit.
V. Handling Procedure for Flask Cultures The flask was seeded with cells grown and completely filled with medium at Cobioer to prevent loss of cells during shipping. 1. Upon receipt visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phasecontrast optics), carefully check for any evidence of microbial contamination. Also check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable). 2. If the cells are still attached, aseptically remove all but 5 to 10 mL of the shipping medium. The shipping medium can be saved for reuse. Incubate the cells at 37°C in a 5% CO2 in air atmosphere until they are ready to be subcultured. 3. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 1000rpm for 5 to 10 minutes. Remove shipping medium and save. Resuspend the pelleted cells in 10 mL of this medium and add to 25 cm2 flask. Incubate at 37°C in a 5% CO2 in air atmosphere until cells are ready to be subcultured.
VI. Subculturing Procedure Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. 1. Remove and discard culture medium. 2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor. 3.Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (depend on the batch of trypsin). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 4.Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 5.Add appropriate aliquots of the cell suspension to new culture vessels. 6.Incubate cultures at 37°C. Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended Medium Renewal: 2 to 3 times per week
VII. Cryopreservation Procedure Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 1. Remove and discard culture medium. 2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor. 3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 1 to 10 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 5. To remove trypsin-EDTA solution, transfer cell suspension to a centrifuge tube and spin at approximately 1000rpm for 5 to10 minutes. 6. Discard supernatant and resuspend cells in cryopreservation medium. 7. Transfer the cells into Cryogenic Vials, 1ml/vial. 8. Frozen the cells in cryogenic container
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