The following hybridoma clones were genetically engineered for expression of Fab′ fragments or murine isotypes: NLDC-145 [American Type Culture Collection (ATCC) HB-290], FGK-45, MIH5, and RMP1-14. Other cell lines used in this study were CT26.WT (ATCC CRL-2638), CT26.PD-L1KO (27 (link)), MC38, and JAWSII (ATCC CRL-11904). For cultivation, NLDC-145, FGK-45, MIH5, RMP1-14, CT26, CT26.PD-L1KO, and MC38 were grown in complete medium formulation consisting of RPMI 1640 medium (11875093, Thermo Fisher Scientific), 2 mM ultraglutamine (BE16-605E/U1, Lonza), 50 μM Gibco 2-mercaptoethanol (2-ME) (21985023, Thermo Fisher Scientific), 1× antibiotic-antimycotic (anti-anti) (15240062, Thermo Fisher Scientific), and 10% heat-inactivated fetal bovine serum (FBS). The JAWSII cell line was cultivated in α–minimum essential medium with ribonucleosides, deoxyribinuuleosides, 4 mM l -glutamine, 1 mM sodium pyruvate, and murine granulocyte-macrophage colony-stimulating factor (5 ng/ml). For expression of PD-L1, MC38 and CT26 were stimulated overnight with recombinant murine IFN-γ (100 ng/ml).
Gibco 2 mercaptoethanol 2 me
Manufactured by Thermo Fisher Scientific
Gibco 2-mercaptoethanol (2-ME) is a reducing agent used in cell culture applications. It is a small organic compound that helps maintain the reducing environment within the cell culture system.
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Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Jawsii, by American Type Culture Collection (1 mentions)
Ct26 wt, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Renca, by American Type Culture Collection (1 mentions)
Phosphate buffered saline (pbs), by Fresenius (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
2 protocols using gibco 2 mercaptoethanol 2 me
1
Generation and Cultivation of Engineered Cell Lines
2
Engineering Hybridoma Cells for PD-L1 Targeting
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Previously, MIH5 hybridoma cells were engineered to express mIgG1, mIgG2asilent or Fab fragments against PD-L1 with a Sortag and a Histag at the C-terminus of the heavy chain(s) (plasmids available at www.addgene.org/ , ID: 124802, 124807 and 124810) [27 (link)]. Other cell lines that were used throughout this study were 4T1 (ATCC CRL-2539) and Renca (ATCC CRL-2947). All cells were cultured in RPMI-1640 (Gibco, 11875-093, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM UltraGlutamine (BE16-605E/U1, Lonza) and 1 × antibiotic–antimycotic (15240-062, Thermo Fisher Scientific). In addition, Hybridoma medium contained 50 μM Gibco 2-mercaptoethanol (2-ME) (21985-023, Thermo Fisher Scientific). Renca cell medium additionally contained 0.1 mM non-essential amino acids (NEAA) (11140-035, Thermo Fisher Scientific) and 1 mM Sodium Pyruvate (Gibco, 11360-070), Thermo Fisher Scientific). (Semi-) adherent cells were washed with phosphate-buffered saline (PBS) (Fresenius Kabi) and detached by incubation with 0.025% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA) in PBS (TE) (Thermo Fisher, R001100) for 10 min. at 37 °C, or by using a cell scraper.
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