Mouse monoclonal MOG antibodies (isotype IgG2B) were produced with the 8-18-C5-specific hybridoma cell line (kindly provided by Christopher Linington, University of Aberdeen, UK) and cultured in Hybridoma-SFM Medium (Gibco; Thermo Fisher Scientific). Supernatant was collected and cells split frequently to ensure optimal growth conditions. Humanized 8-18-C5 was produced by cloning the coding sequence of the variable region of mouse 8-18-C5-specific Kappa-light (8-18-C5-vLC) and heavy F.ab 1 chain (8-18-C5-vHC) (kindly provided by Klaus Dornmair, LMU, Munich, Germany) into the vector pFUSE-CHIg-hG1 (encoding the constant region of the heavy chain) and pFUSE2-CLIg (encoding the constant region of the light chain) (InvivoGen, San Diego, CA, USA), respectively. Vectors, containing 8-18-C5-specific heavy and light chains, were transfected simultaneously into HEK 293FT cells cultured in Freestyle™ Expression Medium (Gibco, Thermo Fisher scientific), using Polyethylenimine (PEI) (1 mg/ml, Sigma-Aldrich) as a transfection agent. Double transfected cells were selected and cultured in growth medium containing Blasticidin (10 μg/ml, Gibco, Thermo Fisher scientific), for pFUSE2-CLIg and Zeocin (400 μg/ml, Invitrogen, Thermo Fisher scientific), for pFUSE-CHIg-hG1. Supernatant was collected and cells split frequently to ensure optimal growth conditions.
Freestyle expression medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
FreeStyle expression medium is a serum-free, chemically defined cell culture medium designed for the transient expression of recombinant proteins in suspension-adapted mammalian cell lines, including HEK293 and CHO cell lines. It supports high-density cell growth and efficient protein production.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Steritop, by Merck Group (2 mentions)
Pei max, by Polysciences (2 mentions)
B16f10 cells, by American Type Culture Collection (2 mentions)
Expi293 cell, by Thermo Fisher Scientific (2 mentions)
Expi293 expression medium, by Thermo Fisher Scientific (2 mentions)
Procho5, by Lonza (2 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Multitron incubation shaker, by Infors (2 mentions)
Huvecs, by Lonza (2 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Hybridoma sfm medium, by Thermo Fisher Scientific (1 mentions)
Pfuse chig hg1, by InvivoGen (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
22 protocols using freestyle expression medium
1
Monoclonal Antibody Production and Humanization
2
Expansion and Transduction of T Cells and Cell Lines
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PBMCS were isolated by Ficoll-Paque (GE Healthcare, cat no. Cytvia 17-1440-03)
from buffy coats obtained from Sanquin Blood Bank). αβT cells were expanded from PBMCs using CD3/CD28 dynabeads (Thermo Fisher scientific, cat no. 40203D) and (1.7 × 103 IU/ml of MACS GMP Recombinant Human interleukin (IL)-7 (Miltenyi Biotec, cat no.130-095-361 ), and 1.5 × 102 IU/ml MACS GMP Recombinant Human IL-15 (Milteny Biotec, cat no. 130-095-762 ). HL60, RPMI 8226, and SSC9 stably expressing GFP-luciferase was generated by a previously described retroviral transduction protocol (30 (link)). The plasmid containing the GFP and luciferase transgenes was kindly provided by Jeanette Leusen (UMC Utrecht, Utrecht, Netherlands). The following cell lines were obtained from ATCC between 2010 and 2018, HL60 (CCL-240), RPMI 8226 (CCL-155), SCC9 (CRL-1629) and Daudi (CCL-213). Freestyle 293-F cells (R790-07) were obtained from Invitrogen. ML-1, HL60, RPMI 8226 and Daudi were cultured in RPMI (Gibco, cat no. 12017599), 10% FCS (Bodinco), 1% Pen/Strep (Invitrogen, cat no. 11548876). Freestyle 293-F in Freestyle expression medium (Gibco, cat no. 10319322). SCC9 in DMEM (Gibco, cat no. 31966047) 10% FCS, 1% Pen/Strep.
from buffy coats obtained from Sanquin Blood Bank). αβT cells were expanded from PBMCs using CD3/CD28 dynabeads (Thermo Fisher scientific, cat no. 40203D) and (1.7 × 103 IU/ml of MACS GMP Recombinant Human interleukin (IL)-7 (Miltenyi Biotec, cat no.
3
Culturing Diverse Cell Lines for Research
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All of the cells were maintained at 37 °C with 5% CO2 unless otherwise noted. Human umbilical vein endothelial cells (HUVECs; Lonza, Allendale, NJ, USA) were cultured in endothelial growth medium‐2 (EGM‐2; Lonza). SNU182 cells (Korean Cell Bank, Seoul, Korea) were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). CFPAC‐1 (ATCC, Manassas, VA, USA) and U87 cells (Korea Cell Line Bank, Seoul, Korea) were cultured in Iscove's modified Dulbecco's medium (Gibco) with the same supplements. HCT116 cells (ATCC) were cultured in McCoy's 5a medium (Corning, Steuben County, NY, USA) with 10% FBS (Corning) and 1× antibiotic/antimycotic (Corning). Bevacizumab‐adapted HCT116 cells (HCT116/Beva) (MD Anderson Cancer Center, Houston, TX, USA) were cultured in McCoy's 5a medium with 10% FBS, 1× antibiotic/antimycotic, and 250 μg·mL−1 bevacizumab (Genentech/Roche, South San Francisco, CA, USA). HEK293F cells were cultured in Freestyle™ expression medium (Gibco) in a humidified Multitron incubation shaker (Infors HT) with 8% CO2.
4
SOSIP Env Protein Production in HEK293F
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SOSIP proteins were expressed in transiently transfected HEK293F suspension cells (Invitrogen, cat no. R79009), maintained in FreeStyle Expression Medium (Gibco). For transfection, HIV-1 Env and furin protease-encoding plasmids mixed in a 4:1 Env to furin ratio (w/w) were incubated with PEImax (Polysciences Europe GmBH, Eppelheim, Germany) in a 3:1 (w/w) PEImax to DNA ratio and then added in the supernatant of cells at a density of 0.8–1.2 million cells/mL. Five to seven days post-transfection, supernatants were harvested, centrifuged, and filtered using Steritops (0.22 µm pore size; Millipore, Amsterdam, The Netherlands) before protein purification.
5
Monocyte-Derived Conditioned Medium Assay
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Human monocyte THP1 cells were cultured in RPMI 1640 medium containing 10% FBS (VivaCell, Shanghai, China) and 1% penicillin‐streptomycin. For CM collection, THP1 cells were plated in a 6‐well plate with FreeStyle expression medium (12 338 026; Gibco, Thermo Fisher Scientific, USA) for 24 h. The CM were collected 24 h later, centrifuged at 15 000 × g for 5 min, and filtered through a 0.22‐mm filter. Human lymphatic endothelial cells were exposed to fresh medium mixed with CM at a ratio of 1:1 (v/v) for the following process.
6
Tumor Cell Culture Protocol
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B16F10 cells were purchased from ATCC (CRL-6475). MC38 cells were a gift from J. Schlom, National Cancer Institute, Bethesda, MD. Apigmented B16F10 cells used for imaging were generated by genetic deletion of Tyrosinase-related-protein-2 (TRP2), referred to as B16F10-Trp2 KO cells94 (link). Tumor cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 10% Fetal Bovine Serum (FBS, Gibco). FreeStyle 293-F cells and Expi293 cells were purchased from Invitrogen (R79007 and A14527, respectively) and cultured in FreeStyle expression medium (Gibco) and Expi293 expression medium (Gibco), respectively. CHO DG44 cells were a gift from David Hacker, EPFL, Lausanne, Switzerland. CHO DG44 cells were cultured in ProCHO5 (Lonza) supplemented with 4 mM L-glutamine, 0.1 mM hypoxanthine, and 16 μM thymidine. Tumor cells were maintained at 37 °C and 5% CO2 and FreeStyle 293-F cells, Expi293 cells, and CHO DG44 cells were maintained at 37 °C and 8% CO2. All cells tested negative for mycoplasma contamination.
7
Protein Expression in HEK293 Cells
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Proteins encoded by the described constructs were expressed either in adherent HEK293T or suspension HEK293F cells, as previously described64 (link),70 (link). HEK293T cells (ATCC, cat. no. CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/mL), and streptomycin (100 µg/mL); and transfected as previously described64 (link). Three days after transfection, HEK293T supernatants were harvested, filtered (0.22 µm pore size) and directly used in supernatant ELISA and BLI assays. Suspension HEK293F cells (Invitrogen, cat. no. R79009) were cultured in FreeStyle Expression Medium (Gibco) and, 5–7 days post-transfection, supernatants were harvested, centrifuged and filtered using 0.22 µm Steritops (Millipore) before protein purification. For transfection, Env- or HA-encoding plasmids were mixed with PEImax (Polysciences, cat. no. 24765) in a 3:1 (w/w) PEImax to DNA ratio. Co-transfection with a furin protease-encoding plasmid was performed in a 4:1 Env to furin ratio only when explicitly indicated.
8
Culturing Pigmented Melanoma and Expression Cells
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B16F10 cells were purchased from ATCC. Apigmented B16F10 cells used for imaging were generated by genetic deletion of Tyrosinase-related-protein-2 (TRP2), referred to as B16F10-Trp2 KO cells (83 (link)). Tumor cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 10% Fetal Bovine Serum (FBS, Gibco). FreeStyle 293-F cells and Expi293 cells were purchased from Invitrogen and cultured in FreeStyle expression medium (Gibco) and Expi293 expression medium (Gibco), respectively. CHO DG44 cells were cultured in ProCHO5 (Lonza) supplemented with 4 mM L-glutamine, 0.1 mM hypoxanthine, and 16 μM thymidine. Tumor cells were maintained at 37°C and 5% CO2 and FreeStyle 293-F cells, Expi293 cells, and CHO DG44 cells were maintained at 37°C and 8% CO2. All cells tested negative for mycoplasma contamination.
9
Culturing Mammalian and Insect Cells
10
Overexpression of IRAP and APN in HEK293-F Cells
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Overexpression of human IRAP and aminopeptidase N (APN) was achieved in Freestyle HEK293‐F Cells (Invitrogen R790‐07). The cells were grown in 80 ml of Freestyle Expression Medium (Invitrogen 12338–018) in 250 ml Erlenmeyer flasks (Corning 431144.40). The flasks were kept at 37 °C, 8 % CO2, 70 % relative humidity and with shaking at 130–135 rpm in an Infors Multitron incubator. The cells were transfected with 1.1 μg IRAP overexpressing plasmid/106 cells30 or 0.7 μg APN overexpressing plasmid17 (kindly provided Dr. Alexandros Nikolau at Vrije Universiteit Brussels, Brussels, Belgium)/106 cells in the presence of 2 μg polyethyleneimine/106 cells (Polyethyleneimine, linear, MW‐25 000, Cat. No. 23966, Polysciences). Prior to overexpression, the plasmids were transfected and propagated in Mach1 E.coli (Invitrogen C8620‐03). The plasmid DNA was prepared using QIAGEN Plasmid Plus Maxi Kit (12963). 48 h post‐transfection, the cells were harvested by centrifugation at 130×g for 3 min. The cells were washed once with PBS and the cell pellets were stored at −20 °C until used for membrane preparations.
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