Human small cell lung cancer cells|100065 |BNCC

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NCI-H446 [H446]

Subculture procedure	resuscitation steps: ① Take out the cryotube from liquid nitrogen or -80 ℃ refrigerator and put it into PE gloves, quickly immerse it in a 37 ℃ water bath, shake the cryotube to accelerate dissolution, and dissolve it completely within 1 minute; ② Add the dissolved cell solution into a centrifuge tube containing 9mL of complete culture medium on a super clean bench, centrifuge at 1000-1200rpm for 5 minutes, discard the supernatant, and resuspend the cells in 1-2mL of complete culture medium. ③ Add the cell suspension into a T25 bottle containing 5-6mL of complete culture medium and incubate in a culture incubator. Cell passage: ① Remove the old culture medium, wash twice with PBS, and add 1-2mL Trypsin (0.25% Trypsin + 0.02% EDTA); ② Under the microscope, observe the digestion process. When the cell edge shrinks and the adhesion becomes loose (you can use a straw to suck up some trypsin and gently blow it onto a certain part of the cell layer. The cell layer can be seen to have fallen off with the naked eye, indicating that digestion is complete. Otherwise, continue digestion). Directly suck out the trypsin, add 5-6mL of complete culture medium, gently blow the cell layer, blow it off, and disperse. ③ Divide the cell suspension into new T25 bottles 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	culture temperature 37 ℃; Gas environment with 5% CO₂ and 95% air;
Safety level	0
morphology	epithelial cell like, short spindle shaped, polygonal, irregular edge
Sharing mode	Public welfare sharing

NCI-H446 [H446] human small cell lung cancer cells

Adherence, epithelial cell-like

No. 100065

Culture:37 ℃,5% CO₂ CM4-1 culture solution

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 4h, 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. CM4-1 culture solution. CM4-1 culture solution: 90% IMDM + 10% FBS. IMDM:IMDM culture solution,

Recovery steps:

(1) Prepare a fresh 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 4-6 days.

Subculture/cryopreservation:remove old medium, and rinse twice with PBS, add 2ml of 0.25%Trypsin+0.02%EDTA, do not shake the culture dish during the period. Observed under the microscope, remove most of the trypsin(with 0.5ml left) when the cells detach. transfer it to the incubator for digestion about 2minute, and take it out.

Passage: terminate digestion with 6ml of CM4-1 culture media, aspirate and dispense into 3~6 culture dishes.

Cryopreservation: terminate digestion with 3ml of cryopreservation solution （90%FBS+10%DMSO), aspirate and dispense into 3 frozen vials, and cryopreserve at -80 ℃ with 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 12 hours, the cell adherence rate ≥ 80.0% in 96hours	adherence is observed in 12 hours, cell adherence rate ≥ 80.0% in 84hours
cell morphology:	adherent, epithelial cell-like	CM4-1 culture medium, adherent, epithelial cell-like, Spindle, polygonal
attached figure:
Conclusion:	good viability, no abnormal cell morphology, qualified

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