Spleen cells were fused with myeloma cells FOX-NY cells (ATCC, VA) using polyethylene glycol, and hybridomas were cultured in a selection medium. ELISAs were performed either with peptain-1, Imject bovine serum albumin (Thermo Scientific, Cat#77110) conjugated peptain-1, scrambled peptain-1 (FEPSVRFSKVDHLVKENDLVK, Peptide 2.0, VA) or human recombinant αB-crystallin to select positive clones by ELISA. Hybridoma clones with high reactivity to peptain-1 and αB-crystallin and no reactivity against scrambled peptide were selected by subcloning. Immunoglobulin G (IgG) subclass was isotyped using Mouse Typer isotyping kit (Bio-Rad, CA, Cat#172–2051). The hybridoma clone that showed strongest immunoreactivity was grown in hybridoma serum free media (Life Technologies, NY, Cat#12045076) for antibody production. The monoclonal antibody was purified on a Protein G-Sepharose column (GE Healthcare, PA) and stored at −80°C until use.
Hybridoma serum free media
Manufactured by Thermo Fisher Scientific
Hybridoma serum free media is a cell culture medium designed for the growth and maintenance of hybridoma cell lines. It is formulated to provide the necessary nutrients and growth factors to support the proliferation of these cells in the absence of serum.
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Lab products found in correlation
3 protocols using hybridoma serum free media
1
Monoclonal Antibody Generation Against αB-Crystallin
2
Cloning and Purification of PVR-like Molecules
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The extracellular domains of CD112R and other PVR-like molecules were cloned and fused into a pMIgV expression vector containing the constant region of mouse IgG2a. Fusion proteins were expressed by transiently transfecting the freestyle HEK293F cells using the polyethylenimine transfection method, and fusion proteins were purified for supernatant using a protein A–Sepharose column according to the manufacturer’s instructions (GE Healthcare).
Mouse anti–human CD112R (clone 2H6; IgG1) was generated from a hybridoma derived from the fusion of SP2 myeloma with B cells from a mouse immunized with human CD112R-Fc. Hybridoma was adapted and cultured in Hybridoma–serum-free media (Life Technologies). Antibodies in supernatant were purified by HiTrap protein G affinity column (GE Healthcare). LEAF purified mouse IgG1 (clone MG1-45) and functional grade human CD112 mAb clone TX31 were purchased from BioLegend. Functional grade human TIGIT mAb (clone MBSA43) was purchased from eBioscience. Human CD226 mAb (clone DX11) was purchased from Abcam. All other antibodies used in flow cytometry were purchased from BD, eBioscience, R&D Systems, or BioLegend.
Mouse anti–human CD112R (clone 2H6; IgG1) was generated from a hybridoma derived from the fusion of SP2 myeloma with B cells from a mouse immunized with human CD112R-Fc. Hybridoma was adapted and cultured in Hybridoma–serum-free media (Life Technologies). Antibodies in supernatant were purified by HiTrap protein G affinity column (GE Healthcare). LEAF purified mouse IgG1 (clone MG1-45) and functional grade human CD112 mAb clone TX31 were purchased from BioLegend. Functional grade human TIGIT mAb (clone MBSA43) was purchased from eBioscience. Human CD226 mAb (clone DX11) was purchased from Abcam. All other antibodies used in flow cytometry were purchased from BD, eBioscience, R&D Systems, or BioLegend.
3
Recombinant TNC Protein Production
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Recombinant full-length TNC (rTNC) was made by transfecting 293T Lenti-X cells with pEE14-HxB.L using jetPRIME transfection reagent (Polyplus Transfection). Cells were incubated in Hybridoma Serum Free Media (Life Technologies) at 37°C for 6 days. Cell supernatant was filtered through a 0.22 μm filter to remove cell debris. Solid ammonium sulfate was added to the cell supernatant to 35% saturation at 4°C and centrifuged to pellet precipitate. The pellet was resuspended in 1XPBS and separated by high performance liquid chromatography (HPLC) using a size exclusion column (Superose 6 30/100 GL; GE Healthcare Life Sciences) equilibrated in 1X PBS buffer. TNC-containing fractions were collected and concentrated using 30K Amicon centrifugal filters (EMD Millipore). Purified TNC was quantified using TNC ELISA and tested against HIV-1 viruses B.YU.2, C.MW965, C.BF1677, C.BF1677, C.BF329NFU2, C.BF942, C.1086, C.DU156.12, and B.MN.3 in a neutralization assay described above.
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