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Hybridoma serum free medium

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Hybridoma serum-free medium is a cell culture medium specifically designed to support the growth and maintenance of hybridoma cells. It is formulated to provide the necessary nutrients and growth factors for the efficient production of monoclonal antibodies without the need for serum supplementation.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Ultra low igg fbs, by Thermo Fisher Scientific (2 mentions) Nutridoma sp, by Roche (2 mentions) Iodoacetamide, by Merck Group (2 mentions) Papain, by Merck Group (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Clonacell hy medium e, by STEMCELL (1 mentions) 0.22 m pore size filter, by Merck Group (1 mentions) 0.22 μm membrane, by Merck Group (1 mentions) Amicon centrifugation filters, by Merck Group (1 mentions) Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions) Hitrap protein g column, by GE Healthcare (1 mentions) Mycoscope, by Genlantis (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Pd 10 desalting column, by GE Healthcare (1 mentions) Hitrap protein g sepharose column, by GE Healthcare (1 mentions) Opi media supplement, by Merck Group (1 mentions)

11 protocols using hybridoma serum free medium

1

Hybridoma Generation and Antibody Production

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Female BALB/c mice were inoculated intramuscularly (i.m.) and boosted 4 weeks later with 6HB peptide (20 µg for each inoculation). Four days after the last inoculation, mice were sacrificed by isoflurane (ISOVA) inhalation and their splenocytes fused to Sp2-0 myeloma cells with polyethylene glycol 4000 (PEG 4000). Hybridomas were selected in ClonaCell-HY Liquid HAT Selection Medium E (StemCell Technologies). Postfusion-specific antibody production in hybridoma supernatants was tested by ELISA using the soluble HPIV3 F protein as described below. Positive cultures were recloned at least twice by limiting dilution using ClonaCell-HY Medium E (StemCell Technologies). The postfusion specificity of supernatant antibodies after cloning was confirmed by ELISA using the soluble HPIV3 F protein and the HRC HRN and 6HB peptides.
Hybridoma cells were maintained in ClonaCell-HY Medium E (StemCell Technologies). For antibody production, flasks were seeded at a confluence of 1 × 106 cells/ml in hybridoma serum-free medium (Gibco). To harvest antibodies, cells and medium were centrifuged at 4°C for 10 min at 1,000 relative centrifugal force (rcf). Supernatant fluid containing antibodies was collected with a 0.22-µm-pore-size filter (Millipore) and stored at −20°C or at 4°C for a maximum of 2 weeks.
Bottom-Tanzer S.F., Rybkina K., Bell J.N., Alabi C.A., Mathieu C., Lu M., Biswas S., Vasquez M., Porotto M., Melero J.A., Más V, & Moscona A. (2019). Inhibiting Human Parainfluenza Virus Infection by Preactivating the Cell Entry Mechanism. mBio, 10(1), e02900-18.
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2

Monoclonal Antibody Production and Purification

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Monoclonal antibodies were produced by culturing hybridoma line 9G11/C7 in hybridoma serum-free medium (Gibco) supplemented with 1% (v/v) ultra-low IgG FBS (Gibco) and 1% (v/v) Nutridoma-SP (Roche) using CELLine disposable bioreactors (Argos Technologies). Medium supernatant was dialyzed against 10 mM Tris/HCl (pH 7.5), 10 mM NaCl (Spectra/Por 6; 10 kDa cutoff) overnight at 4 °C. Precipitates were removed by centrifugation and the supernatant was passed through a 0.2 μm filter and onto a 5 mL HiTrap Q HP column (GE Heathcare) equilibrated in 10 mM Tris/HCl (pH 7.5), 10 mM NaCl. The antibody was eluted from the column using a linear gradient to 10 mM Tris/HCl (pH 7.5), 0.3 M NaCl. Fractions containing the antibody were pooled and digested for 3 h at 37 °C by papain (Worthington) at a 1:50 (w:w) papain:antibody ratio with L-cysteine and ethylenediaminetetraacetic acid (EDTA) added at 10 mM each (Sigma-Aldrich). The digestion was terminated by the addition of 10 mM iodoacetamide (Sigma-Aldrich) for 20 min. The mixture was dialyzed twice against 10 mM Tris/HCl (pH 7.5), 50 mM NaCl overnight at 4 °C and applied to a HiTrap Q HP equilibrated in the same buffer. Fab was collected from the flow-through and concentrated to ~10 mg/mL (Amicon Ultra; 10 kDa cutoff).
Butterwick J.A., del Mármol J., Kim K.H., Kahlson M.A., Rogow J.A., Walz T, & Ruta V. (2018). Cryo-EM structure of the insect olfactory receptor Orco. Nature, 560(7719), 447-452.
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3

Monoclonal Antibody Production and Purification

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Monoclonal antibodies were produced by culturing hybridoma line 9G11/C7 in hybridoma serum-free medium (Gibco) supplemented with 1% (v/v) ultra-low IgG FBS (Gibco) and 1% (v/v) Nutridoma-SP (Roche) using CELLine disposable bioreactors (Argos Technologies). Medium supernatant was dialyzed against 10 mM Tris/HCl (pH 7.5), 10 mM NaCl (Spectra/Por 6; 10 kDa cutoff) overnight at 4 °C. Precipitates were removed by centrifugation and the supernatant was passed through a 0.2 μm filter and onto a 5 mL HiTrap Q HP column (GE Heathcare) equilibrated in 10 mM Tris/HCl (pH 7.5), 10 mM NaCl. The antibody was eluted from the column using a linear gradient to 10 mM Tris/HCl (pH 7.5), 0.3 M NaCl. Fractions containing the antibody were pooled and digested for 3 h at 37 °C by papain (Worthington) at a 1:50 (w:w) papain:antibody ratio with L-cysteine and ethylenediaminetetraacetic acid (EDTA) added at 10 mM each (Sigma-Aldrich). The digestion was terminated by the addition of 10 mM iodoacetamide (Sigma-Aldrich) for 20 min. The mixture was dialyzed twice against 10 mM Tris/HCl (pH 7.5), 50 mM NaCl overnight at 4 °C and applied to a HiTrap Q HP equilibrated in the same buffer. Fab was collected from the flow-through and concentrated to ~10 mg/mL (Amicon Ultra; 10 kDa cutoff).
Butterwick J.A., del Mármol J., Kim K.H., Kahlson M.A., Rogow J.A., Walz T, & Ruta V. (2018). Cryo-EM structure of the insect olfactory receptor Orco. Nature, 560(7719), 447-452.
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4

Purification of Monoclonal Antibodies

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Purification of MAbs was conducted as described previously (30 (link), 34 (link)). Briefly, positive hybridoma clones were expanded and, when at approximately 12 million cells per culture, ∼8 million cells were frozen in a 1-ml cryostock each. Remaining cells were slowly expanded to large culture volumes (∼800 ml each) in hybridoma serum-free medium (Gibco), from which supernatants were harvested via low-speed centrifugation and sterile filtered via 0.22-μM membranes (EMD Millipore). These supernatants were then affinity purified via gravity flow with protein G-linked Sepharose 4 fast flow beads packed into columns (GE Healthcare). After washing the beads with 3 column volumes (∼500 ml) of sterile PBS (pH 7.4), an elution step was carried out with 45 ml of 0.1 M glycine-HCl buffer (pH 2.7). The eluate was then immediately neutralized with 5 ml of 2 M Tris-HCl buffer (pH 10) to bring the overall solution to a pH of approximately 7.0. The MAbs were then buffer exchanged to PBS (pH 7.4) using 30 kDa Amicon centrifugation filters (EMD Millipore) and washed three times with PBS on the membrane. Finally, the concentration of antibody was quantified using a Nanodrop spectrophotometer (Thermo Scientific), measuring absorbance using the protein A280 protocol.
Duehr J., McMahon M., Williamson B., Amanat F., Durbin A., Hawman D.W., Noack D., Uhl S., Tan G.S., Feldmann H, & Krammer F. (2020). Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model. mBio, 11(2), e00028-20.
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5

Producing Anti-Melittin Antibody from Mice

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Antibody production was performed in accordance with protocols approved by the Animal Ethics Committee of the Harry Perkins Institute of Medical Research. Female A/J mice were immunized with honeybee venom collected in Australia. Mice received intraperitoneal injections of 12 μg of venom in Complete Freund’s Adjuvant (Difco), followed by a boost in Incomplete Freund’s Adjuvant on Day 29 and an aqueous boost in PBS at 7 μg/mouse on Day 49. Mice were bled at Day 60, and sera were tested by ELISA. The best responder was boosted with 7 μg of honeybee venom in PBS 4 days prior to fusion. Spleen cells were fused with Sp2/O myeloma cells according to standard procedures82 . Antibody-containing supernatants were screened by ELISA. Hybridoma clone 3B9 was selected for further study. The antibody was produced by growing the hybridoma cells in bioreactors in Hybridoma Serum Free Medium (Gibco). The antibody was purified by protein G-Sepharose chromatography. Purified antibody was dialyzed in PBS (pH 7.3). The antibody was henceforth referred to as the anti-melittin antibody (3B9).
Duffy C., Sorolla A., Wang E., Golden E., Woodward E., Davern K., Ho D., Johnstone E., Pfleger K., Redfern A., Iyer K.S., Baer B, & Blancafort P. (2020). Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer. NPJ Precision Oncology, 4, 24.
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6

Overexpression and Silencing of TP73-AS1

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The full-length TP73-AS1 sequence was synthesized and then sub-cloned into pcDNA3.1 vector (Thermo Fisher Scientific, Inc.) to construct the pcDNA3.1-TP73-AS1 vector. The blank vector was obtained from the Thermo Fisher Scientific, Inc.. Subsequently, the pcDNA3.1-TP73-AS1 vector (2 µg) or the empty vector (2 µg) was transfected into the cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in Hybridoma serum-free medium (Gibco, Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.
The primers (Thermo Fisher Scientific, Inc.) were as follows: TP73-AS1, 5′-TCAGGTTCGTAACGGTGCGTT-3′ (forward) and 5′-TCGTATCTCGCGACTCTTCC-3′ (reverse). The empty pcDNA3.1 vector was used as a negative control. The small interfering RNA (siRNA) sequence for TP73-AS1 was as follows: 5′-CCTGCTGCCTCTCCAAGAGACTGCTATTA-3′. The plasmid of pcDNA/TP73-AS1 was transfected into GES-1 cells (90%) at a density of 0.8×106 cells at a final concentration of 2 µg/ml using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), and were incubated for 48 h.
, & Peng J. (2018). si-TP73-AS1 suppressed proliferation and increased the chemotherapeutic response of GC cells to cisplatin. Oncology Letters, 16(3), 3706-3714.
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7

Purification of Anti-TFF3 Monoclonal Antibodies

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Hybridomas producing anti-TFF3 mAbs were cultured in serum-free condition using a hybridoma serum-free medium (Gibco, Grand Island, NY, USA). The mAbs were purified from culture supernatants by affinity chromatography using HiTrap Protein G columns (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The obtained purified mAbs were checked for their purity and activity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and indirect ELISA, respectively. The purified mAbs were kept at −20 °C.
Khummuang S., Phanphrom W., Laopajon W., Kasinrerk W., Chaiyarit P, & Pata S. (2017). Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva. Biological Procedures Online, 19, 14.
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8

Culturing Diverse Cancer and Immune Cells

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4T1 breast carcinoma cells expressing luc2 (Caliper Life Sciences), mCherry positive 4T1 (kindly provided by Dr. Nicola Aceto, University of Basel) or MMTV-PyMT cells (kindly provided by Dr. David DeNardo, Washington University School of Medicine) were cultured in DMEM supplemented with L-Glutamine and 10% FBS (all GIBCO). AFS98 hybridoma cells (kindly provided by Dr. Richard Stanley, Albert Einstein College of Medicine, NY) were cultured in suspension in Hybridoma serum-free medium (GIBCO). BV2 microglial cells were cultured in RPMI supplemented with L-Glutamine, 10% FBS and 1% antibiotics (all GIBCO). All cell lines were maintained at 37 C in a humified incubator with 5% CO2, and were routinely tested for mycoplasma contamination (Mycoscope, Genlantis).
Tacconi C., Commerford C.D., Dieterich L.C., Schwager S., He Y., Ikenberg K., Friebel E., Becher B., Tugues S, & Detmar M. (2021). CD169+ lymph node macrophages have protective functions in mouse breast cancer metastasis. Cell reports, 35(2).
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9

Antibody Purification from Hybridoma Cells

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Hybridoma cells were cultured with hybridoma serum-free medium (Invitrogen) supplemented with OPI Media Supplement (sigma-aldrich). Antibodies were purified from the conditioned culture media with a HiTrap Protein G-Sepharose column (GE Healthcare Life Sciences). The antibodies are in PBS after going through a PD-10 desalting column (GE Healthcare Life Sciences).
Meng X., Kaever T., Yan B., Traktman P., Zajonc D.M., Peters B., Crotty S, & Xiang Y. (2018). Characterization of murine antibody responses to vaccinia virus envelope protein A14 reveals an immunodominant antigen lacking of effective neutralization targets. Virology, 518, 284-292.
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10

Measuring NK Cell Cytotoxicity

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721.221-ICP47-A1*002 cells were incubated overnight at 26°C with SIV peptides (GenScript and Mimotopes) in Hybridoma-Serum Free Medium (Invitrogen) to stabilize cell surface A1*002-peptide complexes. A GY9 variant with S2A and Y9G substitutions at anchor residues was included as a non-A1*002-binding peptide control. An aliquot of 2 × 105 peptide-pulsed cells were then stained with a PE-conjugated pan-MHC class I specific antibody (clone W6/32; Dako) to verify the surface stabilization of MHC class I molecules. The remaining peptide-pulsed cells were stained with CAM (Invitrogen) at a 1:100 dilution for 1 hour at 26°C. CAM-stained cells were washed and then incubated with Mamu-KIR3DL05+ or -KIR3DL05- NK cells for 4 hours at E:T ratios between 0.5:1 and 10:1 in NKEM at 26°C. The release of CAM into the supernatant was measured using a fluorescent plate reader (excitation 485 nm, absorption 530 nm). Percent specific lysis was calculated as (test release—spontaneous release) / (maximum release—spontaneous release).
Schafer J.L., Ries M., Guha N., Connole M., Colantonio A.D., Wiertz E.J., Wilson N.A., Kaur A, & Evans D.T. (2015). Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides. PLoS Pathogens, 11(9), e1005145.
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