human lymphoendothelial cells|BNCC338560 |BNCC

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human lymphoendothelial 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 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 the T25 flask containing 5-6mL of complete medium and placed in an incubator for culture. Cell subculturing: ① remove the medium, rinse twice with PBS, and add 1-2mL trypsin substitute (TrypLE ™ Express); (2) Observe the digestion under the microscope. When the cell edge shrinks and the adherent is loose (a pipette can be used to suck up some pancreatin substitute to 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 Dispense the cell suspension into a fresh T25 flask according to a 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 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 80%-90%.
Growth conditions	Culture temperature 37 ℃; Atmoshpner 5% CO2 + 95% air;
Growth characteristics	Adherent growth
Storage conditions	Liquid nitrogen
Safety level	1
morphology	Endothelial cells, polygonal, irregular margin, monolayer growth, clean background, no pigment, no vacuoles
Sharing mode	Public welfare sharing

HLEC human lymphoid endothelial cells

Adherence, endothelial cells

Growth:37 ℃,5% CO₂ ECM culture solution

No.: 338560

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 lose of cell viability.

Growth conditions:37 ℃,5% co ₂,ECM culture solution: 500mlECM + 25mlFBS + 5mlECGS + 5mlP/s.

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 4-6 days.

Subculture/cryopreservation:

remove old medium, and rinse twice with PBS, add 2ml 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 1minute, and take it out.

(1)Subculture:terminate digestion with 6ml of CM1-1culturemedia, aspirate and subcultivation ratio of 1:2 is recommended.

(2)Cryopreservation: terminate digestion with 3ml of cryopreservation solution （50% base media+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	adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 140hours	adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 120hours
cell morphology	adherent, endothelial cell	Adherence, endothelial cell, polygon
attached figure
Conclusion	good viability, no abnormal cell morphology, qualified

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