Cytokine Secretion and Viability Assays

Eμ-TCL1 splenocytes, human CLL PBMCs (hCLL), and sorted T-cell subsets from mice and humans were cultured at 1x10⁶/mL for 24 hours in 96 well plates. Concentrations of inhibitors and stimulants are listed in figure legends. Anti-IgM (Jackson ImmunoResearch, West Grove, PA), anti-CD3, anti-CD28 (BioLegend) and MTM (Enzo Life Sciences, Farmingdale, NY) were commercially sourced, while MTM_ox32E was synthesized in house(26 (link)). Secreted cytokines were measured with ELISA MAX Standard Set Mouse or Human kits according to the manufacturer’s protocol (BioLegend). Cellular viability was determined by resuspending cells in cRPMI with 0.1mM resazurin (Sigma Aldrich), incubating for four hours at 37°C, and measuring fluorescence (ex:560nm, em:590nm) on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA)(27 (link)).

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Rivas J.R., Liu Y., Alhakeem S.S., Eckenrode J.M., Marti F., Collard J.P., Zhang Y., Shaaban K.A., Muthusamy N., Hildebrandt G.C., Fleischman R.A., Chen L., Thorson J.S., Leggas M, & Bondada S. (2021). Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia. Leukemia, 35(11), 3188-3200.

Publication 2021

anti-CD3 anti-CD28 ELISA MAX Standard Set Mouse or Human kits resazurin SpectraMax M5 plate reader

Anti cd3 Anti igm Cells Cellular viability Cytokines Devices Elisa Fluorescence Human Inhibitors Mouse Resazurin Stimulants T cell subsets

Corresponding Organization :

Other organizations : University of Kentucky, Markey Cancer Center, The Ohio State University

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Variable analysis

independent variables

Concentrations of inhibitors and stimulants (listed in figure legends)
Anti-IgM (Jackson ImmunoResearch, West Grove, PA)
Anti-CD3
Anti-CD28 (BioLegend)
MTM (Enzo Life Sciences, Farmingdale, NY)
MTM_ox32E (synthesized in-house)

dependent variables

Secreted cytokines (measured with ELISA MAX Standard Set Mouse or Human kits)
Cellular viability (determined by resazurin assay, measured fluorescence)

control variables

Eµ-TCL1 splenocytes, human CLL PBMCs (hCLL), and sorted T-cell subsets from mice and humans cultured at 1x10^6/mL for 24 hours in 96 well plates

controls

Positive control: None explicitly mentioned
Negative control: None explicitly mentioned

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