White blood cells transformed by velvet monkey EBV|338523 |BNCC

Essential Information Certificate Related Products

B95-8

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 6-8mL of complete medium and place the flask in an incubator. Cell passage: ① Suck the culture medium (including suspended cells) into a centrifuge tube, centrifuge at 1000rpm for 5 minutes, and collect the cells; ② Gently rinse T25 bottle twice with PBS, then add 1-2mL pancreatin (0.25% Trypsin+0.02% EDTA); 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 Mix the cells obtained from the above two steps together and divide them into a fresh T25 flask at a ratio of 1:2. Add appropriate complete medium, mix the cell suspension evenly, and culture 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	Epithelial-like，Polygonal，irregular edge，Monolayer adherent growth, partially floating growth
Sharing mode	Public welfare sharing

EBV-transformed white blood cells of B95-8 marinus

Adherent, epithelioid cells

No. 338523

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 CM2-1 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,CM2-1 culture solution. CM2-1 culture solution: 90% RPMI-1640 + 10% FBS. RPMI-1640:1640 culture solution, containing glutamine.

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 6ml 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.

(1)Subculture: terminate digestion with 12ml of CM2-1 culture media, aspirate and dispense into 3~6 culture dishes.

(2)Cryopreservation: terminate digestion with 6ml of cryopreservation solution （90%FBS+10%DMSO), aspirate and dispense into 6 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 12hours, 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, epithelioid cells	CM2-1 culture medium, adherent, epithelioid cells, round, diamond
attached figure
Conclusion	good viability, no abnormal cell morphology, qualified

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