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Media e

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Media E is a cell culture media formulated for the maintenance and expansion of embryonic stem cells. It provides the necessary nutrients and growth factors to support the growth and proliferation of undifferentiated stem cells in vitro.

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Lab products found in correlation

Hybridoma sfm, by Thermo Fisher Scientific (2 mentions) Hitrap mabselectsure columns, by GE Healthcare (2 mentions) Media a, by STEMCELL (1 mentions) Serum free media, by Thermo Fisher Scientific (1 mentions) Protein g column, by GE Healthcare (1 mentions)

3 protocols using media e

1

Isolation and Characterization of Anti-RSV mAb

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25P13 was isolated from the PBMCs of a Nashville Red Cross donor. PBMCs were isolated from human donor blood samples using Ficoll-Histopaque density gradient centrifugation. PBMCs were transformed with Epstein-Barr virus as described previously34 (link). Cells were screened by ELISA for binding to post-fusion RSV F, and positive wells were fused with HMMA2.5 myeloma cells using the previously published protocol34 (link). The 25P13 hybridoma was biologically cloned by single-cell fluorescence-activated sorting. The 25P13 hybridoma was expanded step-wise into 48-well and 12-well plates followed by 75-cm2 flask in Media E (StemCell Technologies). Antibody production was accomplished by expanding the hybridoma to four 225-cm2 cell culture flasks in serum-free medium (Hybridoma-SFM, GIBCO). After 21 days, supernatants were sterile filtered using 0.45 µm pore size filter devices. For antibody purification, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer’s protocol.
Wen X., Mousa J.J., Bates J.T., Lamb R.A., Crowe JE J.r, & Jardetzky T.S. (2017). Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus. Nature microbiology, 2, 16272.
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2

Hybridoma Generation from PBMC B Cells

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Eight days following plating of PBMCs, wells identified to contain positive B cells by ELISA were selected for electrofusion to generate hybridomas as previously described (24 , 25 ). Hybridomas were plated in 384-well plates for HAT selection, and grown for 14 days at 37°C, 5% CO2. Following screening by ELISA, hybridomas were single-cell sorted using a MoFlo Astrios cell sorter using live/dead staining by propidium iodide. The sorted hybridomas were cultured in 25% Media E (StemCell) + 75% Media A (StemCell) for two weeks, then subjected to another round of screening by ELISA. Hybridomas with the highest signal were grown in 250 mL serum-free media (Gibco) for approximately one month. Secreted mAbs were purified using a Protein G column (GE Healthcare) and concentrated for use in downstream assays.
Nagashima K., Dzimianski J.V., Han J., Abbadi N., Gingerich A.D., Royer F., O’Rourke S., Sautto G.A., Ross T.M., Ward A.B., DuBois R.M, & Mousa J.J. (2022). The pre-existing human antibody repertoire to computationally optimized influenza H1 hemagglutinin vaccines. Journal of immunology (Baltimore, Md. : 1950), 209(1), 5-15.
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3

Isolation and Characterization of Anti-RSV mAb

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25P13 was isolated from the PBMCs of a Nashville Red Cross donor. PBMCs were isolated from human donor blood samples using Ficoll-Histopaque density gradient centrifugation. PBMCs were transformed with Epstein-Barr virus as described previously34 (link). Cells were screened by ELISA for binding to post-fusion RSV F, and positive wells were fused with HMMA2.5 myeloma cells using the previously published protocol34 (link). The 25P13 hybridoma was biologically cloned by single-cell fluorescence-activated sorting. The 25P13 hybridoma was expanded step-wise into 48-well and 12-well plates followed by 75-cm2 flask in Media E (StemCell Technologies). Antibody production was accomplished by expanding the hybridoma to four 225-cm2 cell culture flasks in serum-free medium (Hybridoma-SFM, GIBCO). After 21 days, supernatants were sterile filtered using 0.45 µm pore size filter devices. For antibody purification, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer’s protocol.
Wen X., Mousa J.J., Bates J.T., Lamb R.A., Crowe JE J.r, & Jardetzky T.S. (2017). Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus. Nature microbiology, 2, 16272.
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