Mouse t lymphocyte CTLL-2

BNCC number: 359785

Growth characteristics: suspension growth

Growth conditions: 37 ℃,5% CO₂

Complete medium:90%RPMI-1640+10%FBS+100U/mlrmIL-2

Cryopreservation conditions: 50%RPMI-1640＋40%FBS+10%DMSO

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. Under aseptic conditions, transfer the original flask culture solution to a centrifuge tube, centrifuge at 1000-1200rpm/min for 5-10 min, resuspend the cells, transfer the suspended cells to a new T25 flask, add complete culture medium to 10ml, and put it into an incubator for culture. Please strictly follow this instruction, otherwise no reissue service will be provided if cell inactivation is caused.

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, shaken the frozen vial to accelerate dissolution, and it is advisable to dissolve all within 1min; 
(2)Put 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 6-8mL complete medium and put it into an incubator for erection culture.

Cell subculturing: 
(1) Centrifugation: collect cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1mL of culture solution and blow well,

 and dispense the cell suspension into a fresh T25 flask containing 8ml of culture medium according to a ratio of 1:2. 

(2)Half volume solution exchange:  the culture flask is sit for 510 minutes, half of the supernatant medium is gently sucked away,

 the remaining medium with cell precipitation is resuspended and mixed evenly, 

and the cell suspension is dispensed into a fresh T25 flask containing 8ml of medium according to a ratio of 1:2. 

(3) Pay attention to the change of pH value of media and cell density, renew the media regularly (2-3 times a week), and repeat the subculture or cryopreservation when the cell density reaches more than 2 × 10⁶ cells/ml.

Notes:
(1)The cells are density dependent. Initiate the subcultivation in T25 flask when the cell density reaches 80%; A subcultivation ratio of 1:2 is recommended. 
(2)If the cells in sealed culture bottle, put it into the incubator for cultivation after handling, with the cap unscrewed.

Recovery record:  According to the recovery instructions, the results of the cell recovery are reported as follows:

item	quality standard	Recovery record
viability:	suspension growth rate ≥ 80.0% in 168 hours	adherence is observed in 18 hours, cell adherence rate ≥ 80.0% in 144 hours
cell morphology:	suspension, lymphoblast-like	Suspension, lymphoblast-like, round
attached：
conclusion: good viability, no abnormal cell morphology, qualified.