Cell Recovery
Recovery Medium: RPMI 1640+10% FBS+1% P.S+ 2 ng/mL GM-CSF
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 the cell pellet using the recommended complete medium and adjust the viable cell density to 4-6E5 cells/mL. Then dispense the suspension into an appropriate culture flask and initially place the flask in an upright position after thawing.
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 3E6 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+2 ng/mL GM-CSF+3 μg/mL Blasticidin+0.25 μg/mL Puromycin
Approximately 48 - 72 hours after the initial thawing, the cells can be passaged for the first time. After this initial passage, the culture medium can be adjusted to growth medium supplemented with antibiotics.
a) This cell is a human erythroid leukemia cell, lymphoblast, growing in suspension.
b) In the suspension, they appear as large, single, round cells. Cells shed a large accumulation of cytoplasmic granules in the culture, which should not be confused with bacteria!
c) When the cell density reaches 1-1.2E6 cells/mL, perform a 1:2 to 1:3 split, ensuring subculturing every other day. It is essential to perform a full-volume centrifugation and medium replacement during passaging. Do not let the density exceed 1.2E6 cells/mL. It is recommended to use T-25 flasks for subculturing, and you can control the cell density for subculturing by counting.
Subcultivation Ratio: Maintain cultures at a cell concentraion between 4E5 and 6E5 viable cells/mL.
Medium Renewal: Every other day
Notes
a) To minimize the presence of cytoplasmic granules, it is essential to passage the cells every other day when the cell density reaches 1-1.2E6 cells/mL. During passaging, perform a complete centrifugation and replace the culture medium to ensure appropriate cell density and cytokine concentration. Failure to do so may promote the growth of factor-independent subclones.
