BeNa Culture Collection
|Culture medium||DMEM-H complete medium (including 10% FBS):90% DMEM-H + 10% FBS|
|Subculture procedure||Recovery steps: ① The frozen vial is taken out of liquid nitrogen or -80 ℃ refrigerator and put into PE gloves, quickly submersed into a 37 ℃ water bath pan, shaken The frozen vial to accelerate dissolution, and it is advisable to dissolve all within 1min; (2) Add the dissolved cell solution into a centrifuge tube containing 9ml of complete culture medium in an ultra-clean table, centrifuge at 1000-1200rpm for 5min, discard the supernatant, resuspend the cells with 1-2ml of complete media. (3) The cell suspension was added to T25 flask containing 5-6mL complete medium and cultured in an incubator.
cell subculture: ① remove the medium, rinse twice with PBS, and add 1-2mL pancreatin (0.25% Trypsin + 0.02% EDTA); (2) Observe the digestion situation under the microscope. When the cell edge shrinks and the adherent is loose (a pipette can be used to suck up some pancreatin and gently blow somewhere in the cell layer, and the cell layer can be seen to detach with naked eyes, I .e. digestion is completed, otherwise digestion is continued). directly suck out pancreatin, add 5-6mL of complete medium, gently blow the cell layer off. (3) dispense the cell suspension into a fresh T25 flask as a ratio of 1:2, add appropriate complete culture medium, mix the cell suspension evenly, and culture in an incubator.; ④ Pay attention to the change of pH of medium and cell density, renew the medium regularly (2-3 times a week), and repeat the subculture or cryopreservation when the cell density reaches 80%-90%.
|Growth conditions||Culture temperature 37 ℃; Atmosphere 5% CO2 + 95% air;|
|Growth characteristics||Adherent growth|
|Storage conditions||Liquid nitrogen|
|application||This cell line can be used to support mouse embryonic stem cells and induce the growth of pluripotent stem cells (IPS).|
|Sharing mode||Public welfare sharing|
SNL76/7 mouse embryonic fibroblasts
product form: 2ml frozen vial x 2, or T25 flask x 1
Biosafety level: 1, handle in ultra-clear table or safety cabinet
Receiving notice: if any abnormality is found on the day of receiving goods, please contact customer service within 24 hours. if it fails to do so, it will be deemed as good receiving goods. The frozen storage tube shall be stored in a refrigerator at -80 ℃ after receiving the goods. If it is not used for a long time, it shall be transferred to liquid nitrogen storage overnight. T25 form, after receiving the goods, the culture bottle shall be placed in the incubator for rest for 4 hours, and then the cells shall be subjected to routine operation. During recovery, each tube shall not be retained once used up. After recovery, it will be passed on to one generation and can be used normally. Please strictly follow this instruction, otherwise no reissue service will be provided if cell inactivation is caused.
Growth conditions: 37 ℃,5% CO2,90% DMEM-H + 10% FBS. DMEM-H:DMEM high sugar culture solution, containing glutamine and sodium pyruvate.
(1)prepare a new 100mm culture dish containing 12ml of the above culture medium;
(2)remove the frozen vial from liquid nitrogen or refrigerator at -80℃, thaw the vial in 37°C water bath for 1-2 minutes. Transfer the vial to biosafety cabinet for culture as soon as the contents are completely thawed.
(3)draw the solution with a sterile pipette, drip into a new culture dish, and mix it evenly by shaking clockwise;
(4) Put it into incubator (37℃，5%CO2）, change the media overnight, and it will grow up in 2-4 days.
Subculture/cryopreservation: suck out the old culture solution. after PBS cleaning twice, add 2mL(/100mm dish/T25 bottle) of pancreatin (0.25% Trypsin + 0.02% EDTA) and observe under a microscope. during this period, it is forbidden to shake the culture dish. when the cells just fall off, suck out most of the pancreatin, leave about 0.5mL, move to the incubator for digestion, and take out about 1.5min. Subculture is terminated by digestion with 6mL of culture solution, and cells are gently blown evenly, and then subculture can be 1:3. For cryopreservation, 3mL of cryopreservation solution (50% basic medium + 40% FBS + 10% DMSO) is used to terminate digestion, blow evenly, divide into 3 frozen storage tubes, freeze storage at -80 ℃ with a program cooling box, and transfer to liquid nitrogen for storage overnight.
Recovery record: According to the recovery instructions, the results of the cell recovery are reported as follows
|Item||quality standard||recovery record|
|viability:||adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 70hours||adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 66hours|
|cell morphology:||Adherent, fibroblast-like||Adherent, fibroblast-like, short spindle|
|Conclusion:||good viability, and no abnormal cell morphology, qualified|