Cell Recovery
Recovery Medium: RPMI 1640(ATCC)+10% FBS+1% P.S+0.05 mM β-Me
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 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.
Cell Freezing
Freezing Medium: 90% FBS+10% DMSO
a) Centrifuge at 176 x g for 3 minutes to collect cells.
b) Resuspend the cells in pre-chilled cryopreservation medium (90% FBS + 10% DMSO). Given the density-dependent growth characteristics of NCI-H929 cells, it is recommended to increase the freezing density to 5E6 cells/mL and aliquot 1 mL per cryovial.
c) 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(ATCC)+10% FBS+1% P.S+0.05 mM β-Me+0.25 μg/mL Puromycin
After cell thawing, cells should be maintained in recovery medium for the first 1–2 passages. Once the cells have stabilized, the medium can be replaced with growth medium supplemented with antibiotics.
a) This cell line is lymphoblast-like and grows in suspension.
b) After initial recovery, the first passage can typically be performed after approximately 3–4 days. Following two passages, the culture medium can be switched to growth medium supplemented with antibiotics. If passaging is not feasible after 3 days, it is recommended to supplement with recovery medium as appropriate and place the culture flask horizontally.
c) When the cell density reaches 6E5 cells/mL, passage the cells at a ratio of 1:3, and continue passaging every 2–3 days. Do not allow the cell density to exceed 1E6 cells/mL. T25 flasks are recommended for routine subculture.
d) As this is a suspension cell line, the “partial medium replacement” method is recommended during passaging to maintain optimal cell condition. During subculture, fresh growth medium can be added directly to the culture flask, followed by gentle resuspension of the cells. The cell suspension is then transferred into a new T25 flask for continued culture.
Subcultivation Ratio: Maintain cultures at a cell concentraion between 2E5 and 6E5 viable cells/mL.
Medium Renewal: Every 2 to 3 days
Notes
a) Cells are relatively difficult to recover after thawing. When cryopreserved at 5E6 cells/mL, the initial viable cell density after resuscitation may be only around 2E6 cells/mL. Typically, 1–2 weeks of culture adjustment are required for the cells to regain normal growth morphology and density.
b) FBS should be heat-inactivated at 56°C for 30 minutes to inactivate complement proteins and certain viruses, without significantly affecting the activity of most growth factors and cytokines.
Cell Recovery
Recovery Medium: RPMI 1640(ATCC)+10% FBS+1% P.S+0.05 mM β-Me
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 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.
Cell Freezing
Freezing Medium: 90% FBS+10% DMSO
a) Centrifuge at 176 x g for 3 minutes to collect cells.
b) Resuspend the cells in pre-chilled cryopreservation medium (90% FBS + 10% DMSO). Given the density-dependent growth characteristics of NCI-H929 cells, it is recommended to increase the freezing density to 5E6 cells/mL and aliquot 1 mL per cryovial.
c) 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(ATCC)+10% FBS+1% P.S+0.05 mM β-Me+0.25 μg/mL Puromycin
After cell thawing, cells should be maintained in recovery medium for the first 1–2 passages. Once the cells have stabilized, the medium can be replaced with growth medium supplemented with antibiotics.
a) This cell line is lymphoblast-like and grows in suspension.
b) After initial recovery, the first passage can typically be performed after approximately 3–4 days. Following two passages, the culture medium can be switched to growth medium supplemented with antibiotics. If passaging is not feasible after 3 days, it is recommended to supplement with recovery medium as appropriate and place the culture flask horizontally.
c) When the cell density reaches 6E5 cells/mL, passage the cells at a ratio of 1:3, and continue passaging every 2–3 days. Do not allow the cell density to exceed 1E6 cells/mL. T25 flasks are recommended for routine subculture.
d) As this is a suspension cell line, the “partial medium replacement” method is recommended during passaging to maintain optimal cell condition. During subculture, fresh growth medium can be added directly to the culture flask, followed by gentle resuspension of the cells. The cell suspension is then transferred into a new T25 flask for continued culture.
Subcultivation Ratio: Maintain cultures at a cell concentraion between 2E5 and 6E5 viable cells/mL.
Medium Renewal: Every 2 to 3 days
Notes
a) Cells are relatively difficult to recover after thawing. When cryopreserved at 5E6 cells/mL, the initial viable cell density after resuscitation may be only around 2E6 cells/mL. Typically, 1–2 weeks of culture adjustment are required for the cells to regain normal growth morphology and density.
b) FBS should be heat-inactivated at 56°C for 30 minutes to inactivate complement proteins and certain viruses, without significantly affecting the activity of most growth factors and cytokines.
