MDCK33016PF suspension cells (a proprietary cell line developed by Novartis31 (link)–33 (link) and now owned by Seqirus Limited) were maintained at densities between 1–1.5 × 106 cells/ml in 500 ml disposable spinner flasks (Corning, USA) in MDCK 33016 CDM (Lonza, Germany) at 37 °C, 4.5% CO2, shaking at 1 g. Cells were passaged at 3–4 day intervals.
Disposable spinner flasks
Manufactured by Corning
Sourced in United States
Disposable spinner flasks are single-use bioreactor vessels designed for cell culture experiments and small-scale bioprocessing. They provide a controlled environment for growing and maintaining cell lines, enabling researchers to conduct parallel experiments efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti myc antibody, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Freestyle max reagent, by Thermo Fisher Scientific (1 mentions)
Freestyle cho expression medium, by Thermo Fisher Scientific (1 mentions)
Pathhunter cho k1 β arrestin parental cellline, by Eurofins (1 mentions)
Pcmv arms1 pk2 expression vector, by Thermo Fisher Scientific (1 mentions)
Cho s sfm 2, by Thermo Fisher Scientific (1 mentions)
Pmsg elisa kit, by DRG International (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
3 protocols using disposable spinner flasks
1
MDCK33016PF Suspension Cell Maintenance
2
Optimized Recombinant Protein Expression in CHO Cells
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The expression vector, pcDNA3, was purchased from Invitrogen (San Diego, CA,
USA). CHO-K1 cells were obtained from the Japanese Cancer Research Resources
Bank (Tokyo, Japan). Endonucleases were purchased from Boehringer Mannheim (MA,
USA) and Takara (Osaka, Japan). Polymerase chain reaction (PCR) reagents were
from Takara (Japan). Ham’s F-12, CHO-S-SFM II, Geneticin, Lipofectamine
2000, and fetal bovine serum (FBS) were obtained from Gibco BRL (MD, USA). The
QIAprep-Spin plasmid kit was purchased from QIAGEN Inc. (Hilden, Germany).
FreeStyle MAX reagent, FreeStyle CHO expression medium, pCMV-ARMS1-PK2
expression vector, anti-myc antibody, antibiotics, and assay complete medium
were purchased from Invitrogen, the PathHunter CHO-K1 β-arrestin Parental
cell line was obtained from DiscoveRx (San Diego, CA, USA) and disposable
spinner flasks were from Corning Incorporated (NY, USA). PMSG ELISA kit was
obtained from DRG International Inc. (Mountainside, NJ). The cAMP Dynamic 2
immunoassay kit was from Cisbio Bioassay (France).
The oligonucleotides were synthesized by Green Gene Bio (Seoul, Korea). Fetal
bovine serum was from Hyclone laboratories (Utah, USA). Centriplus Centrifugal
Filter Devices were purchased from Amicon Bio separations (MA, USA). All other
reagents used were from Sigma-Aldrich (USA) and Wako Pure Chemicals (Osaka,
Japan).
USA). CHO-K1 cells were obtained from the Japanese Cancer Research Resources
Bank (Tokyo, Japan). Endonucleases were purchased from Boehringer Mannheim (MA,
USA) and Takara (Osaka, Japan). Polymerase chain reaction (PCR) reagents were
from Takara (Japan). Ham’s F-12, CHO-S-SFM II, Geneticin, Lipofectamine
2000, and fetal bovine serum (FBS) were obtained from Gibco BRL (MD, USA). The
QIAprep-Spin plasmid kit was purchased from QIAGEN Inc. (Hilden, Germany).
FreeStyle MAX reagent, FreeStyle CHO expression medium, pCMV-ARMS1-PK2
expression vector, anti-myc antibody, antibiotics, and assay complete medium
were purchased from Invitrogen, the PathHunter CHO-K1 β-arrestin Parental
cell line was obtained from DiscoveRx (San Diego, CA, USA) and disposable
spinner flasks were from Corning Incorporated (NY, USA). PMSG ELISA kit was
obtained from DRG International Inc. (Mountainside, NJ). The cAMP Dynamic 2
immunoassay kit was from Cisbio Bioassay (France).
The oligonucleotides were synthesized by Green Gene Bio (Seoul, Korea). Fetal
bovine serum was from Hyclone laboratories (Utah, USA). Centriplus Centrifugal
Filter Devices were purchased from Amicon Bio separations (MA, USA). All other
reagents used were from Sigma-Aldrich (USA) and Wako Pure Chemicals (Osaka,
Japan).
3
Expansion of Macrophage Progenitors
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Freshly harvested macrophage progenitors were collected and cultured over several weeks in suspension cultures. To this end, disposable spinner flasks (Corning, Somerville, MA, USA) were placed on magnetic stirrers (30 rpm) in a humidified incubator (37 °C, 5% CO2). Cells were cultured in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 2 mM Glutamax, 1% penicillin/streptomycin, 50 ug/mL mercaptoethanol, M-CSF (100 ng/mL), and IL3 (25 ng/mL). To accumulate sufficient cells for screening purposes, several harvests of one “factory” were accumulated in the suspension cultures over the course of several weeks. Cell number was adjusted to 0.5–2 × 106 cells/mL, 50% medium exchange was performed twice a week, cells were resuspended and counted, and quality control was performed by assessing marker gene expressions (CD68, Ki67, CD11b, and CD14) by flow cytometry.
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