CHO-S suspension cells (Life Technologies, Thermo Scientific, Rockford, IL) were grown as previously described (Grav et al., 2015 (link)). In summary, cells were grown in CD CHO medium supplemented with 8mM L-glutamine and 2 μL/mL anti-clumping agent (Life Technologies). Cells were expanded in Corning vent cap shake flasks (Sigma-Aldrich, St.Louis, MO) in a humidified incubator at 120 rpm (25 mm orbit), 37°C and 5% CO2. Viable cell densities were measured using the NucleoCounter NC-200 Cell Counter (ChemoMetec, Allerod, Denmark) and cells were passed into fresh medium every two to three days with seeding densities at 3–5×105 cells/mL.
Cho s suspension cells
Manufactured by Thermo Fisher Scientific
CHO-S suspension cells are a mammalian cell line derived from Chinese Hamster Ovary (CHO) cells, designed for use in suspension cell culture applications. The cells are adapted to grow in suspension in a serum-free media, which simplifies the culturing process and allows for scalable production of recombinant proteins and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Anti clumping agent, by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Cd cho medium, by Thermo Fisher Scientific (3 mentions)
Nucleocounter nc 200 cell counter, by ChemoMetec (2 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Dulbecco s modified eagle s medium, by Lonza (1 mentions)
Amaxa cell line nucleofector kit 5 system, by Lonza (1 mentions)
Erlenmeyer flask, by Corning (1 mentions)
Nucleofector 2b device, by Lonza (1 mentions)
Freestyle tm max reagent, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Lonza (1 mentions)
Pmaxgfp vector, by Lonza (1 mentions)
Opti pro sfm medium, by Thermo Fisher Scientific (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
7 protocols using cho s suspension cells
1
Culturing Suspension CHO-S Cells
2
Engineered Antibody Fragment Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO adherent cells (CHO) L761H were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% foetal calf serum. CHO-S suspension cells (Life Technologies) were grown in CD CHO chemically defined medium. All HCs contained either the human IgG1 or IgG4 CH1,2,3 domains fused to either mouse or humanised VH domains with specificity for A33 [13 (link)], TNF-α [14 (link)], CD20 [15 (link)], CD25 (Zenapax) [16 (link)], CD25 (Simulect) [17 (link)] and CD11a [18 (link)]. The variable domain from an in-house humanised mouse antibody (277) was also used [19 (link)]. Variable domains were synthesised by DNA 2.0. All IgG4 HCs contain an S225P mutation (i.e. –ESKYGPPCPSCP– to –ESKYGPPCPPCP–), which reduces the amount of HC : LC dimers formed [20 (link)]. Additional mutations introduced by site-directed mutagenesis included C127S in the A33 Fab fragment CH1 domain and P151A in the A33 HC CH1 domain as indicated. The Fab HC constructs were prepared by PCR using primers complementary to the 5′ sequence and to the junction between the CH1 domain and the hinge region. The 3′-primer was also designed to add a V5-tag and a stop codon. The final construct is depicted in Figure 4B .
3
Bacterial and Mammalian Cell Culture Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All standard cloning and plasmid propagation was performed in Escherichia coli strain DH5α, which was grown in standard Luria Broth (LB) medium supplemented with 100 µg/ml ampicillin.
Human U-2-OS osteosarcoma cells and HEK293 cells both obtained from ATCC were grown in Dulbecco's modified Eagle's medium (Lonza, Vervier, Belgium) supplemented with 10% (v/v) FBS (Lonza), 100 µg/ml streptomycin and 100 U/ml penicillin (Lonza). CHO-S suspension cells obtained from Life Technologies were grown in CD CHO medium (Gibco, Life Technologies) supplemented with 8 mM L-Glutamine (Gibco, Life Technologies) and 1∶500 Anti-Clumping Agent (Gibco, Life Technologies).
Human U-2-OS osteosarcoma cells and HEK293 cells both obtained from ATCC were grown in Dulbecco's modified Eagle's medium (Lonza, Vervier, Belgium) supplemented with 10% (v/v) FBS (Lonza), 100 µg/ml streptomycin and 100 U/ml penicillin (Lonza). CHO-S suspension cells obtained from Life Technologies were grown in CD CHO medium (Gibco, Life Technologies) supplemented with 8 mM L-Glutamine (Gibco, Life Technologies) and 1∶500 Anti-Clumping Agent (Gibco, Life Technologies).
4
CHO-S Cell Culture Expansion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO-S suspension cells (Life Technologies) were grown in CD CHO medium supplemented with 8 mM L-glutamine and 2 μL/mL anti-clumping agent (Life Technologies). Cells were expanded in Corning vent cap shake flasks (Sigma-Aldrich, St. Louis, MO) in a humidified incubator at 120 rpm (25 mm orbit), 37 °C, and 5% CO2.
5
Stable CHO Cell Line Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO-S suspension cells (Life Technologies) were grown in CD CHO medium (Life Technologies) supplemented with glutamine synthetase expression medium supplement (GSEM; Sigma Aldrich, St. Louis, MO) and 2 μL/mL anti-clumping agent (Life Technologies).
Cells were maintained in 125 mL Erlenmeyer flasks (Corning Inc., Acton, MA), incubated at 37°C, 5% CO2 at 120 rpm (25 mm shaking amplitude) and passaged every 2-3 days. In order to establish a stable etanercept-expressing polyclonal cell line, 4.5 x 10 6 cells were transfected with 5.4 µg of plasmid DNA using Amaxa Cell Line Nucleofector Kit V system (Lonza, Basel, Switzerland) and program U-024 on a Nucleofector 2b device (Lonza), according to the manufacturer's protocol. Cells were then seeded at 4.5 x 10 5 cells/mL in a 125 mL shake flask and three days after transfection, selection for GS expression was initiated by adding 25 µM MSX (Sigma Aldrich; Cat. M5379). After 14 days of selection, cells were subjected to an increased selection pressure (50 µM MSX).
Cells were maintained in 125 mL Erlenmeyer flasks (Corning Inc., Acton, MA), incubated at 37°C, 5% CO2 at 120 rpm (25 mm shaking amplitude) and passaged every 2-3 days. In order to establish a stable etanercept-expressing polyclonal cell line, 4.5 x 10 6 cells were transfected with 5.4 µg of plasmid DNA using Amaxa Cell Line Nucleofector Kit V system (Lonza, Basel, Switzerland) and program U-024 on a Nucleofector 2b device (Lonza), according to the manufacturer's protocol. Cells were then seeded at 4.5 x 10 5 cells/mL in a 125 mL shake flask and three days after transfection, selection for GS expression was initiated by adding 25 µM MSX (Sigma Aldrich; Cat. M5379). After 14 days of selection, cells were subjected to an increased selection pressure (50 µM MSX).
6
Transient Expression of Recombinant Proteins in CHO-S Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO-S suspension cells (Life Technologies, Thermo Scientific, Rockford, IL ) were grown in CD CHO medium (#10743029, Life Technologies) supplemented with 8 mM L-glutamine (#LONZ17-605F, Lonza Group AG, Basel, Switzerland) and 2 µL/mL anti-clumping agent (Life Technologies). Cells were expanded in Corning vent cap shake flasks (Sigma-Aldrich, St. Louis, MO) in a humidified incubator at 120 rpm (25 mm orbit), 37°C, and 5% CO2. Transfection was performed essentially as previously described [13] . In brief, 3x10 7 (recombinant human erythropoietin; rEPO) or 5x10 7 (empty vector and α1AT) cells were transfected using FreeStyle TM MAX Reagent (Life Technologies) in 30 mL (EPO) or 50 mL (empty vector and α1AT) complete CD CHO medium without anti-clumping agent according to manufacturer's instructions. Transfected cells were incubated in Corning vent cap shake flasks (Sigma-Aldrich) at 120 rpm (25 mm orbit), 37°C, and 5% CO2. 3 hours post-transfection, anti-clumping agent was added to reach a 2 µL/mL final concentration. Viable cell density (VCD) and viability were measured every day (day 0 -3) and supernatant samples were obtained from day 1 to day 3.
7
CRISPR Transfection of CHO-S Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO-S suspension cells (Life Technologies, Carlsbad, CA) were cultivated in CD CHO medium supplemented with 8 mM L-glutamine and 1 μL/mL anti-clumping agent (Life Technologies). Cells were incubated in a humidified incubator at 120 rpm, 37 °C and 5% CO2.
Cell passaging was conducted every two to three days at 3 x 10 5 cells/mL after measuring viable cell densities (VCDs) and viabilities with the NucleoCounter NC-200 Cell Counter (ChemoMetec, Allerod, Denmark). One day prior transfection with CRISPR reagents, anti-clumping agent was removed by centrifugation and 5 -6 x 10 5 cells/mL were seeded in a six multi-well well plate (BD Biosciences, San Jose, CA) for each transfection. At the day of transfection each sample was seeded at 1 x 10 6 cells/mL and a total DNA load of 3.5 μg was transfected with FuGENE ® HD transfection reagent (Promega, Madison, WI) and OptiPRO SFM medium (Life Technologies) according to the manufacturer´s recommendations. The GFP_2A_Cas9 / sgRNA plasmid ratios for each sample are presented in Table S3. To measure transfection efficiency, pmaxGFP ® vector (Lonza, Basel, Switzerland) transfection was performed. Cells were harvested for fluorescence-activated cell sorting (FACS) 48 h after transfection.
Cell passaging was conducted every two to three days at 3 x 10 5 cells/mL after measuring viable cell densities (VCDs) and viabilities with the NucleoCounter NC-200 Cell Counter (ChemoMetec, Allerod, Denmark). One day prior transfection with CRISPR reagents, anti-clumping agent was removed by centrifugation and 5 -6 x 10 5 cells/mL were seeded in a six multi-well well plate (BD Biosciences, San Jose, CA) for each transfection. At the day of transfection each sample was seeded at 1 x 10 6 cells/mL and a total DNA load of 3.5 μg was transfected with FuGENE ® HD transfection reagent (Promega, Madison, WI) and OptiPRO SFM medium (Life Technologies) according to the manufacturer´s recommendations. The GFP_2A_Cas9 / sgRNA plasmid ratios for each sample are presented in Table S3. To measure transfection efficiency, pmaxGFP ® vector (Lonza, Basel, Switzerland) transfection was performed. Cells were harvested for fluorescence-activated cell sorting (FACS) 48 h after transfection.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!