Clone mopc 173

Jurkat cells were cultured in RPMI1640 medium supplemented with 10% FBS and activated with 10 ng/mL phorbol myristate acetate (PMA; Thermo Fisher Scientific) or with Dynabeads Human T-activator CD3/CD28 (Thermo Fisher Scientific). After activation, the cells were collected, stained with a cell viability dye (Ghost V450, TONOBO), and the following allophycocyanin (APC)-conjugated antibodies: anti-human CD69 (BioLegend, clone FN50), or with isotype control (BioLegend, clone MOPC-173 and clone MOPC-21; isotype 1 and isotype 2, respectively) and analyzed using flow cytometry.
Human peripheral blood mononuclear cells (PBMC) and healthy mouse splenocytes were cultured in RPMI1640 medium supplemented with 10% FBS at a density of 1 × 10⁶ cells/mL. Cells were activated with 25 μL of CD3/CD28 beads per 1 × 10⁶ cells (Gibco, Dynabeads human or mouse T-activator) and recombinant human IL2 (BioLegend). Subsequently, the cells were collected and stained with a cell viability dye (Ghost UV780, TONOBO), blocked with TruStain FcX (BioLegend, human or anti-mouse CD16/32, clone 93), and stained with CD3 (BioLegend, anti-human, clone HIT3a or anti-mouse, clone 17A2) and CD69 (BioLegend, anti-human, clone FN50 or anti-mouse, clone H1.2F3) for flow cytometric analysis.

Antibodies to the surface epitopes CD14 (clone HCD14, Pacific Blue-conjugated; and clone M5E2, Brilliant Violet 510-conjugated), CD16 (clone 3G8, PE-conjugated), TLR2 (clone TL2.1, PE-conjugated) and TLR4 (clone HTA125, Brilliant Violet 421-conjugated) as well as isotype controls (clone MOPC-173, clone MOPC-21, clone RTK2071) were purchased from BioLegend. For intracellular cytokine staining, antibodies to TNF-α (clone MAb11, PerCP/Cy5-5-conjugated), IL-1β (clone H1b-98, Alexa Fluor 647-conjugated), IL-8 (clone E8N1, Alexa Fluor 488-conjugated) and IL-10 (clone JES3-9D7, PE/Cy7-conjugated) were also obtained from BioLegend. Fixable viability stain (eFluor® 780, APC-H7-conjugated) was purchased from eBioScience (San Diego, CA).

Whole blood (WB) was cultured using a volume of 0.5–1 ml blood per culture condition in round bottom 5 ml polypropylene tubes. For stimulation the samples were treated with SLA (10 µg/ml). Control samples were treated with PBS. In some assays PHA (10 µg/ml) or Staphylococcus enterotoxin B, SEB, (5 µg/ml) was used as positive controls (not shown). Samples were incubated for 37°C in the presence of 5% CO₂ for 24 hours if not otherwise indicated in figure text.
To block HLA-TCR interaction 20 µg/ml anti-HLA-DR, clone 243, or isotype control IgG2a, clone MOPC-173, both ultra-LEAF purified (BioLegend, US) were added to the cultures simultaneously with antigen.