BeNa Culture Collection
|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 ℃ refrigerator and put it into PE gloves, quickly submerse into a 37 ℃ water bath, shake the frozen vial to accelerate dissolution, and it is advisable to dissolve all within 1min; (2) Add the dissolved cell solution into a centrifuge tube containing 9ml of complete culture medium in an ultra-clean table, centrifuge at 1000-1200rpm for 3-5min, discard the supernatant, resuspend the cells with 1-2ml of complete media. ③The cell suspension was then added to the T25 flask containing 6-7mL of complete medium and placed in an incubator for culture. The culture flask is recommended to be cultured uprightly.
cell subculture: ① centrifugation: collect cells, centrifuge at 1000rpm for 5min, 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 medium exchange method: half-volume medium exchange method can be selected; Please gently suck half of the supernatant culture medium, resuspend and mix the remaining culture medium with cell precipitation, and divide the cell suspension into a fresh T25 flask containing 8mL culture 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|
|morphology||lymphoblastoid, round, irregular, margin irregular, single cell, monolayer growth, clean background, no pigment, no vacuoles|
|Separation substrate||Macrophages; J2 retrovirus transfection; C57BL/6|
|Sharing mode||Public welfare sharing|
Ana-1 mouse macrophage
Product format: 2ml frozen vial x 2, or centrifuge tube 15mL
Biosafety level: 1 , handle in ultra-clean 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,CM2-1 culture solution. CM2-1 culture solution: 90% RPMI-1640 + 10% FBS. RPMI-1640:1640 culture solution, containing glutamine.
(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 3-5 days.
Subculture: the suspended cells were gently beaten and evenly distributed into 2-4 fresh culture medium dishes. After being shaken well, the cells were put into the incubator for culture
Cryopreservation: The suspended cells were transferred to the centrifuge tube for (110g, 3min) centrifugation，after centrifugationterminate 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||suspension growth rate ≥ 80.0% in 108hours||inoculation with 20% cell solution, suspension growth rate reaches 30.0% in 18hours, and 80.0% in 90hours|
|cell morphology||suspension, macrophage-like||in CM2-1 culture solution, suspended, macrophage-like, irregularly round, slightly adherent|
|conclusion||good viability, and no abnormal cell morphology, qualified|