Cd69 antibody

All lymphocytes, pre- and post-selection, were analyzed using flow cytometry and the following antibodies: anti-CD45 (AF700; clone HI300), anti-CD3 (PE, FITC or PE-Cy7; clone OKT3), anti-CD8 (PE or BV510; clone HIT8a), anti-CD4 (BV421 or BV785; clone OKT4), anti-CD27 (BV785; clone 323), anti-CD14 (PC7; clone HCD14), anti-CD56 (APC; clone MEM-188), anti-EGFR (PE; clone AY13), TCRαβ (APC-Fire750; clone IP26) and anti-CD19 (BV421; clone HIB19) (all from Biolegend). To determine T cell activation state, T cells were stained with CD69 antibody (APC-Fire750; clone FN50) and CD25 antibody (BV650; clone BC96) (Biolegend) at indicated time points after initial stimulation. For live/dead discrimination propidium iodide (Sigma) was used. CAR expression has been monitored using an anti-idiotype antibody specific for the CD19-directed CAR, an in-house developed antibody. Cell associated fluorescence was analyzed by flow cytometry using either CytoFLEX or CytoFLEX LX flow cytometers (Beckman Coulter), unless indicated otherwise.
Live nucleated cells were enumerated pre- and post-selection (including fractions and downstream culture) using either NuceloCounter NC-3000 (Chemotec) or XN-350 Cell Counter (Sysmex) according to manufacturers’ instructions.

Spleens were excised from C57BL/6 mice and placed in complete RPMI 1640 media supplemented with heat inactivated 10% fetal bovine serum, 10mM L-glutamine, 10mM HEPES, 50µM β-mercaptoethanol, and 100µg/ml penicillin/streptomycin. Tissues were homogenized into single-cell suspensions and subjected to red blood cell lysis. Cells were plated in a 96-well plate in complete media at 1x10⁶ cells per well for 24 hours at 37°C and 5% CO₂ with or without SEB-stimulation (1µg/ml) and with vehicle, I3C or DIM (100µM). Vehicle for all compounds was dimethyl sulfoxide (DMSO), with a total volume of never exceeding 0.5% DMSO in complete medium per well. Cells were harvested and counted after 24 hours. To assess activation, cells collected from in vitro cultures were stained with CD69 antibody purchased from Biolegend (San Diego, CA) for flow cytometry analysis.

Cell surface marker expression was analyzed by flow cytometry. To this end, Fc receptors were blocked (anti-CD16/CD32; eBioscience), cells were stained with fluorescently labeled CD11b (BioLegend) and F4/80 (eBioscience) antibodies or CD69 antibody (BioLegend), and fixed with 2% PFA/PBS. CD11b, F4/80, and CD69 expression were analyzed in the FACS Canto-II flow cytometer using the FACS Diva software (BD Biosiences). The FlowJo software was then used for further data analysis.
Phagocytic activity was analyzed by incubating 0.5 × 10⁶ mature macrophages with PE-labeled beads at an MOI of 10. After 2 or 20 h, adherent cells were washed once with PBS and the uptake of beads was analyzed by flow cytometry (FACS Canto-II), gating on PE+ cells. Further analysis of the number of phagocytosed beads was carried out using the FlowJo software by calculating the median fluorescence intensity of PE+ macrophages.