Human peripheral blood B lymphocytes|BNCC358130 |BNCC

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Human peripheral blood B lymphocytes

Culture medium	RPMI-1640 complete medium: 90% RPMI-1640 + 10% FBS
Subculture procedure	Recovery steps: ① Take out the frozen vial from liquid nitrogen or -80 ℃ and put it into PE gloves, quickly subtract into a water bath pot at 37 ℃, shake the frozen vial to accelerate dissolution, and completely dissolve within 1 minute. (2) Add the dissolved cell solution to 9ml in the ultra-clean table; In the centrifuge tube of complete culture medium, centrifuge at 1000-1200rpm/min for 5 minutes, discard the supernatant, resuspend the cells with 1-2ml of complete media. ③The cell suspension was then added to a T25 flask containing 6-8mL complete medium and placed in an incubator for upright cultivation. Cell subculturing: ① Centrifugation: collect cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1-2mL 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 liquid medium exchange: after the culture flask is allowed to stand for 5-10 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 divided into a fresh T25 flask containing 8ml of medium according to a ratio of 1:2. (3) Pay attention to the change of pH of medium and cell density, renew the medium regularly (2-3 times a week), and repeat the subculture or cryopreservation when the cell density reaches more than 2 × 106 cells/ml.
Growth conditions	Culture temperature 37 ℃; Atmosphere 5% CO2 + 95% air;
Growth characteristics	Suspension growth
Storage conditions	Liquid nitrogen
Type	Suspension growth
Safety level	2
morphology	Lymphoblast-like, round, single cell, few clubbed, clean background, no vacuoles
Separation substrate	Peripheral blood
Sharing mode	Public welfare sharing

IM-9 human peripheral blood B lymphocytes

BNCC number: 358130

Cell name: IM-9 human peripheral blood B lymphocyte

Growth characteristics: Suspension growth

Growth conditions: 37 ℃,5% CO₂ complete medium: 90% RPMI-1640 + 10% FBS

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. 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.

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 6-8 ml complete medium and cultured in an incubator.

Cell subculturing:
(1 ) Centrifugation: collect cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1-2mL 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 5-10 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 128 hours	adherence is observed in 18 hours, cell adherence rate ≥ 80.0% in 114 hours
cell morphology:	suspension, lymphoblast	Suspension, lymphoblast, round, partially clustered growth
attached：
Conclusion:	good viability, and no abnormal cell morphology, qualified