Human mammary epithelial cells|337734 |BNCC

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MCF-10A

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 Frozen 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 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; 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 5-6 milliliters of culture medium containing 10% FBS to terminate digestion, gently blow the cell layer, blow it off, and disperse it. ③ Centrifuge the cell suspension at 1000rpm for 3 minutes, discard the supernatant, resuspend the cells in 1-2mL of complete culture medium, and then add the cell suspension in a 1:2 ratio to a T25 bottle containing 5-6mL of complete culture medium. Mix the cell suspension evenly and culture it in an 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	Epithelial-like，Polygonal，irregular edge
Sharing mode	Public welfare sharing

MCF-10A human mammary epithelial cells

Adherent, epithelioid cells

No.: 337734

Product format: 2ml frozen vial x 2, or T25 flask x 1

Biosafety level: 1 , handle in ultra-clean table or safety cabinet

Culture:37 ℃,5% CO₂ CM30-1 culture solution

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 lose of cell viability.

Growth conditions:37 ℃,5% CO₂,CM30-1 culture solution. CM30-1 culture solution: 500ml MEpiCM +5ml MEpiCGS.

Recovery steps:
(1)Prepare a new 100mm culture dish containing 12ml of the above culture medium;
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.
(2)Draw the solution with a sterile pipette, drip into a new culture dish, and mix it evenly by shaking clockwise;
(3)Put it into incubator (37℃，5%CO₂）, change the media overnight, and it will grow up in 2-4 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 4minute, and take it out.

(1)subculture: terminate digestion with 6ml of complete media supplemented with10%FBS, add 3ml of CM30-1 media after centrifuge, aspirate the cells, and dispense into 3-6 culture vessel.

(2) 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 18 hours, the cell adherence rate ≥ 80.0% in 107hours	adherence is observed in 12 hours, the cell adherence rate ≥ 80.0% in 100hours
cell morphology	adherent, epithelioid cells	in CM30-1 culture solution, adherent, epithelioid cells, round, short spindle
attached figure
conclusion	good viability, and no abnormal cell morphology, qualified

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