Cell Recovery
Recovery Medium: RPMI 1640+10% FBS+1% P.S+1% Glutamax+1% Sodium Pyruvate
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.
a) Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 - 3 minutes).
b) Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
c) Transfer the vial contents to a centrifuge tube containing 5.0 mL complete culture medium. And spin at approximately 176 x g for 5 minutes.Discard supernatant.
d) Resuspend cell pellet with the recommended complete medium. And dispense the suspension into 1 - 2 T-25 culture flasks.
e) Incubate the culture at 37°C in a suitable incubator. A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.
Freezing Medium: 90% FBS+10% DMSO
a) Centrifuge at 176 x g for 3 minutes to collect cells.
b) Resuspend the cells in pre-cooled freezing medium and adjust the cell density to 5E6 cells/mL.
c) Aliquot 1 mL into each vial.
d) Place the vial in a controlled-rate freezing container and store at -80°C for at least 1 day, then transfer to liquid nitrogen as soon as possible.
Cell passage
Growth medium: RPMI 1640+10% FBS+1% P.S+1% Glutamax+1% Sodium Pyruvate+0.25 μg/mL Puromycin
After this initial passage, the culture medium can be adjusted to growth medium supplemented with antibiotics.
a) This is a mixed cell culture; cells grow both as a lightly attached monolayer and in suspension.
b) When the cell density reaches 1.5 - 2E6 cells/mL, continue passaging and ensure that the density does not exceed 2E6 cells/mL.
c) Cultures can be maintained by adding fresh medium. Alternatively, subcultures can be prepared by scraping the adherent cells into the medium containing the floating cells, collecting the cells by centrifugation, resuspending the cell pellet in fresh medium and dispensing into new flasks.
Subcultivation Ratio: Maintain cultures at a cell concentraion between 3E5 and 1E6 viable cells/mL.
Medium Renewal: Every 2 to 3 days
Notes
a) After thawing, cells need to be cultured for about two weeks to restore their normal growth state.
b) Ensure proper nutrition, and if not handling them, make sure to add an appropriate amount of medium every other day.
