Three cell lines, CHO-hCD137, CHO-mFcγRIIb, and LLC1/OVA/hGPC3, were established previously [10] (link). CHO-hCD137, which is a CHO-DG44-based cell line transfected with an expression vector, expresses human CD137 [10] (link). CHO-mFcγRIIb is a murine FcγRII-overexpressing CHO cell line expressing murine FcγRII derived from CHO-DG44 [10] (link). LLC1/OVA/hGPC3 expresses chicken ovalbumin (OVA) and human GPC3 and was established from the murine cell line LLC1 [10] (link). CHO-mFcγRIIb and CHO-hCD137 were maintained in CHO-S-SFM II medium (Thermo Fisher Scientific) supplemented with 1% HT supplement (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific) and 500 μg/mL G418 (Thermo Fisher Scientific) in a humidified incubator maintained at 37 °C with 5% CO2. LLC1/OVA/hGPC3 was maintained in D-MEM high glucose medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 500 µg/mL G418 (Nacalai Tesque), and 1 mg/mL Zeocin (Thermo Fisher Scientific) in a humidified incubator maintained at 37 °C with 5% CO2.
Cho s sfm 2 medium
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
CHO-S-SFM II medium is a serum-free, animal component-free cell culture medium specifically formulated for the growth and maintenance of Chinese Hamster Ovary (CHO) cells in suspension culture. It is designed to support the optimal growth and viability of CHO-S cells.
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Lab products found in correlation
Protein g sepharose, by GE Healthcare (3 mentions)
Cho s cells, by Thermo Fisher Scientific (2 mentions)
Zeocin, by InvivoGen (2 mentions)
Pcag neo, by Fujifilm (2 mentions)
Pcag ble, by Fujifilm (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ht supplement, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose medium, by Merck Group (1 mentions)
Pcdna3.1 zeo, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Expicho expression system, by Thermo Fisher Scientific (1 mentions)
Syncropatch 384i, by Nanion Technologies (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Patchcontrol 384, by Nanion Technologies (1 mentions)
Geneticin, by InvivoGen (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
Nbactiv1 media, by Transnetyx (1 mentions)
Mea plate, by Axion Biosystems (1 mentions)
Laminin, by Merck Group (1 mentions)
6 protocols using cho s sfm 2 medium
1
Establishment and Maintenance of Cell Lines
2
Generation of Chimeric Anti-Human CAR Antibody
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A mouse anti‐human CAR mAb, mu6G10A, was developed as described previously.14 Generation of mouse‐human chimeric anti‐human CAR (ch6G10A) was previously described.18 Briefly, the appropriate VH and VL cDNAs of mu6G10A and CH and CL of human IgG1 were subcloned into pcDNA3.3/Neo or pcDNA3.1/Zeo vectors (Thermo Fisher Scientific Inc.), respectively. Antibody expression vectors were transfected into CHO‐S cells (Thermo Fisher Scientific Inc.) using Lipofectamine 2000 reagent (Thermo Fisher Scientific Inc.). Stable transfectants of CHO‐S/ch6G10A were selected by culturing the transfectants in medium containing 1 mg/mL G418 (Nacalai Tesque, Inc.) and 0.5 mg/mL zeocin (InvivoGen). Stable transfectants were cultured for 14 days in CHO‐S‐SFM II medium (Thermo Fisher Scientific Inc.), and then ch6G10A immunoglobulin was purified from the culture supernatant using Protein G Sepharose (GE Healthcare United Kingdom, Ltd). Purity of ch6G10A was evaluated by SDS‐PAGE and Coomassie Brilliant Blue staining.
3
Generation of Anti-PODXL Monoclonal Antibodies
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PcMab-47, a mouse anti-PODXL mAb (IgG1, kappa), was developed as previously described [17 (link)]. Mouse IgG was purchased from Sigma-Aldrich. To generate 47-mG2a, appropriate VH and VL cDNAs of mouse PcMab-47 and CH and CL of Mouse IgG2a were subcloned into pCAG-Ble and pCAG-Neo vectors (Wako Pure Chemical Industries, Osaka, Japan), respectively. Antibody expression vectors were transfected into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher Scientific). To generate 47-mG2a-f, antibody expression vectors were also transfected into PDIS-5 cells using the ExpiCHO Expression System. Stable transfectants of chPcMab-47 cells were established previously [34 (link)]. CHO-S/chPcMab-47 cells were cultivated in CHO-S-SFM II medium (Thermo Fisher Scientific). PcMab-47, 47-mG2a, 47-mG2a-f, and chPcMab-47 were purified using Protein G-Sepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Goat polyclonal anti-human PODXL and mouse monoclonal anti-human PODXL mAb (53D11) were purchased from R&D systems, Inc. (Minneapolis, MN) and Medical & Biological Laboratories Co., Ltd. (MBL; Nagoya, Japan), respectively.
4
Automated Patch Clamp of Transfected Cells
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Automated patch clamp recording was conducted with a SyncroPatch 384i (Nanion Technologies) using planar borosilicate glass medium-resistance chips in a 384-well microtiter plate format with one or four holes per well and Nanion Standard solutions (for their composition, see Table S1 ). Transfected cells were dissociated using TrypLE Express, diluted with CHO-S-SFM-II medium (both from Thermo Fisher) and resuspended in external physiological solution (Nanion Technologies) at 1 × 105 to 4 × 105 cells ml−1. Each well was filled with 30 μl Chip Fill solution, to which 20 μl of the cell suspension was added. Seal formation was enhanced by the addition of 40 μl of NMDG (N-methyl-d -glucamine) 60 solution with 10 mM CaCl2 (final concentration). After capturing the cells, 50 μl of the external solution was replaced with 40 μl of NMDG 60 solution, and 40 μl of the mixture was removed. For data acquisition and analysis, respectively, PatchControl384 and DataControl384 software v. 1.9.0 were used (both Nanion Technologies). Illumination was provided with Luxeon Z blue LEDs, LXZ1-PB01 (470 ± 20 nm), controlled by custom-built software.
5
Generation of Anti-hPDPN Chimeric Antibody
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LpMab‐2, a mouse anti‐hPDPN mAb (IgG1, kappa), was developed as previously described 29 . Human IgG was purchased from Beckman Coulter, Inc. (Fullerton, CA). To generate human–mouse chimeric anti‐hPDPN (chLpMab‐2), appropriate VH and VL cDNAs of mouse LpMab‐2 and CH and CL of human IgG1 were subcloned into pCAG‐Ble and pCAG‐Neo vectors (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. CHO‐S/fucosyltransferase 8 (FUT8)‐KO (PDIS‐5) cell lines were generated by transfecting CRISPR/Cas9 plasmids that targeted FUT8 (Target ID: HS0000547010; Sigma‐Aldrich Corp., St. Louis, MO) into CHO‐S cells (Thermo Fisher Scientific Inc., Waltham, MA) using a Gene Pulser Xcell electroporation system. PDIS‐5 cells were screened using Aleuria aurantia lectin. Antibody expression vectors were transfected into PDIS‐5 using the Lipofectamine LTX reagent (Thermo Fisher Scientific Inc.). Stable transfectants of PDIS‐5/chLpMab‐2 were selected by cultivating the transfectants in a medium containing 0.5 mg/mL of both geneticin and zeocin (InvivoGen, San Diego, CA). PDIS‐5/chLpMab‐2 cells were cultivated in CHO‐S‐SFM II medium (Thermo Fisher Scientific Inc.). chLpMab‐2 was purified using Protein G‐Sepharose (GE healthcare Bio‐Sciences, Pittsburgh, PA).
6
Rat Cortical Neuron Culture Protocol
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Rat cortical cultures were prepared from E18 rat embryos (BrainBits, Springfield, IL) dissociated by trituration after papain digestion as per the manufacturer's protocol. The cells were resuspended in the NBActiv1 media (BrainBits) supplemented with 5% fetal bovine serum and 20 µg/mL laminin (Sigma-Aldrich, Natick, MA). Neurons were seeded at 80,000 cells/well on a 48-well microelectrode array (MEA) plate (Axion BioSystems, Atlanta, GA) covered with adhesion promoting molecules (PEI). For experiments on the SyncroPatch 384PE, dissociated neurons were plated in 100 mm of poly-d-lysine-coated dishes at 4 × 10E6 to 6 × 10E6 cells/dish. Cells were fed every 3-4 days by replacing approximately half of the media with serumfree media. On the day of the experiment, cells were harvested as described previously [13] [14] [15] with trypsin-EDTA solution (PCS-999-003, ATCC, Manassas, VA) for 5-10 min at 37 °C and subsequently neutralized in serum-free CHO-S-SFM II medium (Thermo Fisher, Bedford, MA). Cells were then centrifuged and resuspended in chip fill solution supplemented with 3 mM MgCl 2 and gently pipetted 10 times to break up cell clumps; cells were then prepared at 100,000 cells/mL for SyncroPatch recording.
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