Mouse fibroblast|100314 |BNCC

Essential Information References Certificate Related Products

L929[NCTC clone 929]

Subculture procedure	Resuscitation steps: ① Remove the frozen vial from liquid nitrogen or -80 ℃ refrigerator and place it in PE gloves. Quickly immerse it in a 37 ℃ water bath, shake the vial to accelerate dissolution, and dissolve it completely within 1 minute; ② Add the dissolved cell solution into a centrifuge tube containing 9mL of complete medium on a ultra-clean bench, centrifuge at 1000-1200rpm for 5 minutes, discard the supernatant, and resuspend the cells in 1-2mL of complete medium. ③ Add the cell suspension into a T25 flask containing 5-6mL of complete medium and place the flask in an incubator. Cell passage: 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 pasteur 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 Divide the cell suspension into a fresh T25 flask at a ratio of 1:2, add appropriate complete culture medium, mix the cell suspension evenly, and culture it in the incubator.; ④ Pay attention to changes in the pH value of the culture medium and cell density, change the medium regularly (2-3 times a week), and repeat the passage operation or cryopreservation when the cell density reaches 80% -90%.
Growth conditions	37°C; 5% CO₂ + 95% air
Safety level	0
morphology	Fibroblast-like，Polygonal，irregular edge，Monolayer adherent growth
Sharing mode	Public welfare sharing

L929 mouse fibroblasts

Adherent, fibroblast-like

No. 100314

Product format: 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 receipt, please contact the customer service within 24 hours. If it is overdue, it is deemed that the cells are well. For the frozen cells, it shall be stored in the refrigerator at -80 ℃ upon arrival. If they are not used for a long time, they shall be transferred to liquid nitrogen for storage overnight. Recovered cells in T25 culture flask, upon receipt, put the culture flask in the incubator for 2-3h, and then carry out handling procedure. During recovery, each vial shall be used up once and shall not be retained. After recovery, the cells can be passed on to the next generation and can be used normally. Please operate in strict accordance with this instruction, otherwise the replacement of cells are not be available in case of loss of cell viability.

Growth conditions:37 ℃,5% CO₂,CM8-1 culture solution. CM8-1 culture solution: 90% EMEM + 10% FBS. EMEM:EMEM culture solution.

Recovery steps:

① 1 new 100mm plate containing 12mL of the above culture solution;

② Take out the frozen storage tube from liquid nitrogen or -80 ℃, take a water bath at 37 ℃ for 1~2min, and move it into the safety cabinet for recovery as soon as possible after it is completely dissolved.

③ Use a sterile straw to suck the dissolved liquid into the new plate and shake well clockwise;

④ Put it into an incubator (37 ℃,5% CO₂), change the liquid overnight, and fill it in 3-5 days.

Subculture/cryopreservation: Aspirate the old culture medium, wash twice with PBS, add 2mL (/100mm dish/T25 bottle) trypsin (0.25%Trypsin+0.02%EDTA), observe under the microscope, do not shake the culture dish during this period , When the cells just fall off, aspirate most of the trypsin, leave about 0.5 mL, move it to the incubator for digestion, and take it out for about 2 minutes. Use 6 mL of culture medium to stop digestion, gently pipette evenly, and then pass 1:3; for cryopreservation, stop digestion with 3 mL of freezing medium (50% basal medium + 40% FBS + 10% DMSO), pipette evenly, Divide into 3 cryovials and freeze at -80°C in a programmed cooling box.

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 96 hours	adherence is observed in 18 hours, cell adherence rate ≥ 80.0% in 90 hours
cell morphology:	Adherent, fibroblast-like	CM8-1 culture medium, adherent, fibroblast-like, round, polygonal
attached figure:
Conclusion:	good viability, and no abnormal cell morphology, qualified

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