Human brain microvascular endothelial cells|BNCC350771 |BNCC

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Human brain microvascular endothelial cells

Culture medium	Endothelial cell special medium (ECM):500mlECM 25mlFBS 5mlECGS 5mlP/S
Subculture procedure	Recovery steps: ① Take out the frozen vial from liquid nitrogen or -80 ℃ and put it into PE gloves, quickly submerse into a 37 ℃ water bath pot, shake the frozen vial to accelerate dissolution, and it is advisable to dissolve all within 1 minute; (2) Add the dissolved cell liquid into a centrifuge tube containing 9ml of complete culture medium in an ultra-clean table, 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 the T25 flask containing 5-6mL of complete medium and placed in an incubator for culture. Cell subculturing: ① remove the medium, add 1-2mL) pancreatin after PBS cleaning twice; (2) Observe the digestion under the microscope, when the cell edge shrinks and the adherent is loose (use a pipette to suck up some pancreatin and gently blow somewhere in the cell layer, the cell layer can be seen to detach with the naked eye, I .e. digestion is completed, otherwise digestion is continued), directly suck out pancreatin, add 5-6ml of complete culture medium, and gently blow the cell layer, blow down the cell layer and blow it away. ③ Dispense the cell suspension into a fresh T25 flask as the ratio of 1:2, add appropriate complete culture medium, mix the cell suspension evenly, and culture in an incubator. ④ 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 80%-90%.
Growth conditions	37 ℃;5% CO2 + 95% air;
Growth characteristics	Adherent growth
Storage conditions	Liquid nitrogen
Type	Adherent growth
Safety level	1
morphology	Endothelial cells, irregular shape, irregular margin, monolayer adherent growth, no vacuoles
Separation substrate	human, brain microvascular; endothelial cell
application	HBMEC is characterized by immunofluorescence with vWF/factor VIII and CD31 (PECAM)-specific antibodies.
Sharing mode	Public welfare sharing

Human brain microvascular endothelial cells & nbsp;

HBMEC

No. 350771

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

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,ECM culture solution: 500mlECM + 25mlFBS + 5mlECGS + 5mlP/S. ECM: Special Medium for ECM Endothelial Cells.

Recovery steps:
(1)prepare a new 100mm culture dish containing 12ml of the above culture medium;
（2）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.
(3)draw the solution with a sterile pipette, drip into a new culture dish, and mix it evenly by shaking clockwise;
(4) Put it into incubator (37℃，5%CO2）, change the media overnight, and it will grow up in 4-6 days.

Subculture/cryopreservation: remove the medium, rinse twice with PBS , and add 2mL pancreatic enzyme substitute(TrypLE ™ Express); observe under a microscope, do not shake the culture dish during this period, when the cells detach, suck out most of the pancreatin with about 0.5ml remained, move to the incubator for digestion, and take out in about 2min. For subculture, digestion is terminated by 6mL of culture solution, and subculture at 1:2. For cryopreservation, terminate the digestion with 3mL of cryopreservation solution ( 50% base medium+40%FBS+10%DMSO ), blow evenly, dispense into 3 frozen vials, and freeze at -80 ℃ with a program 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 120hours	adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 110hours
cell morphology:	Adherent, Endothelial Cell	Adherent, Endothelial Cells, Irregular
attached figure:
Conclusion:	good viability, no abnormal cell morphology, qualified