Characterization of 4-1BB and 4-1BBL Expression

The African green monkey kidney cell line Vero 10–87 was obtained from WHO (world health organization, Geneva, Switzerland). The Jurkat cell line (JE6.1) and the mouse thymoma cell line BW5147 were derived from in house stocks and cultured in RPMI1640 supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) (from Sigma-Aldrich, St. Louis, MO). For transfection, HEK293T cells (kindly provided by A. Carmo, Porto, Portugal) were used. Mycoplasma contaminations were detected by using a recently described reporter system based on a human monocytic THP-1 cell line [31 (link)]. The following antibodies were purchased from Biolegend (San Diego, CA): 4-1BB (CD137)-PE (4B4-1), m4-1BB (CD137)-PE (17B5), 4-1BBL (CD137L)-PE (5F4), m4-1BBL (CD137L)-PE (TKS-1), CD14-APC (M5E2), CD4-BV421 (OKT4), CD8-PerCP (HIT8α), CD56-PE (5.1H11), CD56-BV510 (5.1H11), CD25-PeCy7 (M-A251). A DyLight-649-conjugated goat-anti-mouse IgG(H + L) antibody from Jackson ImmunoResearch (West Grove,PA) was used for anti-CD3 single chain fragment detection. The Strep-tag was detected by using the monoclonal mouse NWSHPQFEK-Tag (biotin) antibody (GeneScript, Leiden, The Netherlands) in conjunction with a Streptavidin-PE (Biolegend, San Diego, CA). The agonistic anti-human 4-1BB antibody urelumab (human, IgG4) was obtained from Creative Biolabs (Shirley, NY).

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Battin C., De Sousa Linhares A., Leitner J., Grossmann A., Lupinek D., Izadi S., Castilho A., Waidhofer-Söllner P., Grabmeier-Pfistershammer K., Stritzker J, & Steinberger P. (2023). Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents. Cancer Immunology, Immunotherapy, 72(9), 3029-3043.

Publication 2023

RPMI1640 FBS penicillin streptomycin CD25-PeCy7 (M-A251). Streptavidin-PE

4 1bbl African green monkey Agonistic Anti antibody Anti cd3 Anti igg Antibodies Antibody Biotin Cd137 Cd25 Cell line Cells Goat Human Igg4 Jurkat cell Kidney Monocytic Mouse Mycoplasma Penicillin Strep tag Streptavidin Streptomycin Thp 1 cell line Thymoma Transfection Urelumab

Corresponding Organization : Medical University of Vienna

Other organizations : BOKU University

Top 5 similar protocols

Variable analysis

independent variables

Mycoplasma contaminations detected by using a recently described reporter system based on a human monocytic THP-1 cell line

dependent variables

Mycoplasma contamination

control variables

African green monkey kidney cell line Vero 10–87
Jurkat cell line (JE6.1)
Mouse thymoma cell line BW5147
HEK293T cells
RPMI1640 supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL)
Antibodies from Biolegend (San Diego, CA): 4-1BB (CD137)-PE (4B4-1), m4-1BB (CD137)-PE (17B5), 4-1BBL (CD137L)-PE (5F4), m4-1BBL (CD137L)-PE (TKS-1), CD14-APC (M5E2), CD4-BV421 (OKT4), CD8-PerCP (HIT8α), CD56-PE (5.1H11), CD56-BV510 (5.1H11), CD25-PeCy7 (M-A251)
DyLight-649-conjugated goat-anti-mouse IgG(H + L) antibody from Jackson ImmunoResearch (West Grove,PA)
Strep-tag detected by using the monoclonal mouse NWSHPQFEK-Tag (biotin) antibody (GeneScript, Leiden, The Netherlands) in conjunction with a Streptavidin-PE (Biolegend, San Diego, CA)
Agonistic anti-human 4-1BB antibody urelumab (human, IgG4) obtained from Creative Biolabs (Shirley, NY)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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