PGT125, PGT126, PGT128 and PGT130 Fabs were expressed in 293S (ATCC) or FreeStyle 293F cells (Life Technologies) grown in suspension. The cells were transfected at a 1:1 ratio of plasmids encoding the respective antibodies’ light and heavy chains (truncated at AspH234). Supernatants were harvested six days after transfection, and cell debris was removed by centrifugation. Antibody was purified from the clarified supernatant using an anti-human lambda affinity matrix (CaptureSelect LC-lambda (hu); Thermo Scientific) equilibrated in PBS. Fab fragments were eluted with 0.1 M glycine, pH 3, and the eluate neutralized with concentrated PBS. Recombinant IgGs were expressed in FreeStyle 293F cells and purified on a protein A resin (Thermo Scientific) as described elsewhere.[13 (link)]
Freestyle 293 f cells
Manufactured by Thermo Fisher Scientific
Sourced in United States, France, Norway
FreeStyle 293-F cells are a human embryonic kidney (HEK) cell line derived from the 293 cell line. They are designed for use in transient protein expression and production in suspension culture. FreeStyle 293-F cells are capable of growing in serum-free media and can be transfected efficiently with various transfection reagents.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle 293 expression medium, by Thermo Fisher Scientific (82 mentions)
Expi293f cells, by Thermo Fisher Scientific (28 mentions)
Freestyle 293f medium, by Thermo Fisher Scientific (23 mentions)
Expifectamine 293 transfection reagent, by Thermo Fisher Scientific (19 mentions)
Hek293t cell, by American Type Culture Collection (16 mentions)
0.22 μm filter, by Thermo Fisher Scientific (16 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Hitrap, by Cytiva (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (13 mentions)
Nickel affinity column, by Thermo Fisher Scientific (13 mentions)
293fectin, by Thermo Fisher Scientific (12 mentions)
Polyethylenimine (pei), by Polysciences (12 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (12 mentions)
Freestyle 293 f, by Thermo Fisher Scientific (11 mentions)
Glutamax, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Superose 6 10 300 column, by GE Healthcare (8 mentions)
293fectin transfection reagent, by Thermo Fisher Scientific (7 mentions)
370 protocols using freestyle 293 f cells
1
Recombinant Antibody Expression and Purification
2
Clec9A and RNF41 Protein Expression and Purification
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Clec9A ecto-domains and control proteins fused to a FLAG-tag and a biotinylation consensus sequence were expressed in mammalian Freestyle 293 F cells (Thermo Fisher) using Freestyle Max (Thermo Fisher) and purified using anti-FLAG M2 affinity gel (Sigma-Aldrich) and size exclusion chromatography (SEC) as previously described (Zhang et al., 2012 (link)). cDNA constructs encoding full-length FLAG-tagged Clec9A were subcloned into either pEF-Bos or pcDNA3.1+ (Invitrogen) for mammalian expression as previously described (Zhang et al., 2012 (link)). cDNA constructs encoding full-length and truncated versions of mouse and human RNF41 for mammalian expression (RNF41 full-length: 1-MGYD…VEEI-317; RNF41- ΔRING: 72-MRNM…VEEI-317) were subcloned into pcDNA3.1+ (Invitrogen). cDNA constructs encoding RNF41 for bacterial expression (RNF41-RBCC: 1-MGYD…VEEI-317; RNF41-BCC: 72-MRNM…VEEI-317; RNF41-CC: 135-IKHL…VEEI-317; RNF41-C: 169-DIQL…VEEI-317; RNF41-RBC: 1-MGYD…RAIR-181) were cloned into a modified pGEX-2T vector (GE Healthcare). His-tagged and untagged RNF41 proteins were expressed in mammalian Freestyle 293 F cells using 293Fectin (Thermo Fisher). GST-fusion RNF41 recombinant proteins were expressed in bacterial BL21 DE3 E. coli (Promega) and purified using Glutathione-Sepharose resin (GE Healthcare) and SEC.
3
Stable Antibody Expression in Freestyle-293F Cells
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Freestyle-293F cells (R79007; Thermo Fisher) cells were grown in a 6-well culture plate at a density of 2 × 106 cells/well/2 ml media. 24 h later, pAb20-hCHIgG1 and pAb20-hCK plasmids separately expressing heavy and light chain of antibodies were transiently co-transfected into Freestyle-293F cells (R79007; Thermo Fisher) using purefection reagent (LV750A-1; System Bio) followed by manufacturer protocol. 48-hours post-transfection, cells were selected in presence of hygromycin (200 μg/ml) and blasticidin (10 μg/ml) for the selection of stable clones expressing heavy and light chains. The selected individual colonies were observed for 2 weeks and proceeded with trypsinization and limited dilution method implied for the generation of single-cell monoclones. Furthermore, monoclones showing the highest titer in ELISA were selected for further characterization.
The stable cells were then cultured in a shaker incubator run at 120 rpm with conditions of 8% CO2 at 37 °C. After one week, the supernatants consisting of secreted antibodies were collected and trapped by protein G Sepharose (GE Healthcare). The bound antibodies on the Sepharose were eluted and concentrated using Centricon Plus-70 (051555; Millipore). Successively, purified antibodies were utilized in the following binding and neutralization analysis.
The stable cells were then cultured in a shaker incubator run at 120 rpm with conditions of 8% CO2 at 37 °C. After one week, the supernatants consisting of secreted antibodies were collected and trapped by protein G Sepharose (GE Healthcare). The bound antibodies on the Sepharose were eluted and concentrated using Centricon Plus-70 (051555; Millipore). Successively, purified antibodies were utilized in the following binding and neutralization analysis.
4
Recombinant RSV F protein production
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Plasmids of antibodies, including AM22, D25, MOTA, MPE8, 101F and 12A12, were kindly donated by Jason S. McLellan (Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, TX, USA) and expressed using FreeStyleTM 293-F cells (Gibco) as previous described [24 (link)].
The soluble post-F protein was constructed as described by McLellan et al. [25 (link)], and the soluble pre-F protein SC-TM was constructed as described by Anders Krarup et al. [12 (link)]. The soluble pre-F and post-F proteins were expressed in FreeStyleTM 293-F cells (Gibco), and purified by a two-step protocol applying a cation exchange chromatography followed by SEC. The eluate was concentrated and the protein was further purified on a Superdex200 column (GE Healthcare). A reduced SDS–PAGE analysis was used to determine purity of the final protein preparation. The identity of the prefusion conformation and postfusion conformation was verified using ELISA with RSV antibody panel (Supplementary Figure S1 ).
The soluble post-F protein was constructed as described by McLellan et al. [25 (link)], and the soluble pre-F protein SC-TM was constructed as described by Anders Krarup et al. [12 (link)]. The soluble pre-F and post-F proteins were expressed in FreeStyleTM 293-F cells (Gibco), and purified by a two-step protocol applying a cation exchange chromatography followed by SEC. The eluate was concentrated and the protein was further purified on a Superdex200 column (GE Healthcare). A reduced SDS–PAGE analysis was used to determine purity of the final protein preparation. The identity of the prefusion conformation and postfusion conformation was verified using ELISA with RSV antibody panel (
5
Murine Pancreatic Organoid Culture
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FACs sorted cells from murine tissue were resuspended in >90% v/v Matrigel (Corning) and grown in murine pancreatic organoid medium (MPOM) containing advanced DMEM/F12 (Gibco) containing 10 mmol/L HEPES (Gibco), 1X GlutaMAX (Gibco), 1x penicillin/streptomycin (Gibco), 5% v/v Rspo2-Fc conditioned medium (harvested from transiently transfected FreestyleTM-293F cells, Thermo Fisher), 5% v/v Noggin-conditioned medium (harvested from Noggin-expressing 239 cells obtained from Foundation Hubrecht Organoid Technology (HUB), Hubrecht Institute, Uterecht, The Netherlands), 10 mmol/L nicotinamide, 1% v/v B-27 supplement without vitamin A, 1 mmol/L N-acetyl-L-cysteine, 100 ng/mL rh FGF-10, 50 ng/mL rh EGF, 10 nmol/L rh [Leu15]-gastrin I, and 3 µmol/L prostaglandin E2. Following passaging, the organoids were cultured in MPOM with 10 µM Y-27632 (Sigma) and 5 µM GSK-3 inhibitor (Sigma) for the first three days, followed by culturing in normal MPOM for regular maintenance, as described previously [62 (link)].
6
Henipavirus Propagation and Titration in Cell Lines
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Vero cells (catalog no. GDC0029), Vero E6 cells (catalog no. GDC0146), and 293T cells (catalog no. GDC0187) were obtained from the China Center for Type Culture Collection (CCTCC). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin–streptomycin (Beyotime) in a 37 °C, 5% CO2 atmosphere. FreeStyleTM 293-F cells (cat#R79007) were obtained from Thermo Fisher and maintained in FreestyleTM 293 expression medium (Thermo Fisher) at 37 °C in an 8% CO2 atmosphere.
Nipah virus Malaysia strain (CSTR: 16533.06.IVCAS 6.7489), Nipah virus Bangladesh strain (CSTR: 16533.06.IVCAS 6.7488), and Hendra virus (CSTR: 16533.06.IVCAS 6.7487) were obtained from the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Science. Each virus was passaged in Vero cells. All processes in this study involving authentic henipavirus were performed at the Wuhan National Biosafety Laboratory under biosafety level 4 (BSL-4) conditions. Virus titers were determined using 50% tissue culture infectious dose (TCID50) method. The median lethal dose (LD50) was calculated by Reed and Muench.
Nipah virus Malaysia strain (CSTR: 16533.06.IVCAS 6.7489), Nipah virus Bangladesh strain (CSTR: 16533.06.IVCAS 6.7488), and Hendra virus (CSTR: 16533.06.IVCAS 6.7487) were obtained from the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Science. Each virus was passaged in Vero cells. All processes in this study involving authentic henipavirus were performed at the Wuhan National Biosafety Laboratory under biosafety level 4 (BSL-4) conditions. Virus titers were determined using 50% tissue culture infectious dose (TCID50) method. The median lethal dose (LD50) was calculated by Reed and Muench.
7
Stable Cell Line Generation for PD-L1 and LAG-3
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CHO-S cells were purchased from Invitrogen (Carlsbad, USA) and transfected to stably express human PD-L1 according to the manufacturer’s instructions using the Freedom CHO-S Kit (Invitrogen, USA). FreeStyleTM 293-F cells were purchased from Thermo Fisher Scientific and transfected to stably express human LAG-3. The human PD-L1 knock-in MC38 cell line was constructed by Nanjing Galaxy Biopharma Co., Ltd. Briefly, mouse PD-L1 was replaced by human PD-L1 expression frame based on CRISPR/Cas9 technology.
8
Mammalian Cell Culture Protocols
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Cell lines used in this study were obtained from ThermoFisher Scientific (FreeStyleTM 293-F Cells, Expi293FTM Cells and ExpiCHO-STM).
9
Production and Characterization of HCVpp
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The human embryonic kidney epithelial cell line 293T with stably knocked out CD81 (the main cell surface receptor for HCV entry), kindly donated by Joe Grove, University College London, UK, was used to produce GT1a and GT3a HCVpp. The lack of CD81 on these cells prevents attachment of HCVpp to their cell surface, thereby allowing accumulation of the HCVpp to the culture supernatant leading to increased yield. FreeStyleTM 293 F cells (Thermo Fisher) were used to produce recombinant GT1a and GT3a HCV envelope proteins. The human hepatocyte cell line HuH‐7.5 with high CD81 expression obtained from Charles M. Rice, Rockefeller University, was infected with the GT1a or GT3a HCVpp and used to determine neutralizing activities of antibodies in the serum of patients infected with the corresponding viral genotype. The monocytic cell line THP‐1 (ATCC 202 TIB) was used as effector cells for Fc‐receptor mediated‐ADCP of patient plasma opsonized microbeads by flow cytometry as described.
28 (link)
28 (link)
10
Culturing HEK293T and HEK293F Cells
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HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with GlutaMAX and 10% (v/v) fetal bovine serum (Invitrogen, Inc.) at 37°C, 10% CO2 and 95% humidity. HEK293F cells (Free StyleTM 293-F Cells, Thermo-Fischer Scientific) were grown in FreeStyle 293 Expression Medium (Invitrogen, Inc.) in flasks on a rotary shaker (120 rpm) at 37°C, 8% CO2 and 70% humidity.
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