Indo 1am dye

CAR T cells and ChTCR T cells were harvested and washed once with phosphate buffer saline (PBS). Cells were stained with anti-human CD45 antibody (clone HI30), so that each receptor was stained with a unique CD45 fluorescent barcode (single or double stain with APC-CD45 (Biolegend, 304012), PE-CD45 (BD, 555483), PerCp-Cy-5.5-CD45 (BD, 564105), FITC-CD45 (Biolegend, 304006), BUV805-CD45 (BD, 612891)). Cells were washed three times, pooled 10⁷ cells total. Cells were stained with 5μM indo-1AM dye (Invitrogen, I1223) in calcium stain buffer (phenol-free RPMI, 1%FBS, 0.5 mM probenecid (Sigma, P8761–100G), 10 mM HEPES) at 37° for 45 minutes. Cells were then washed twice with calcium stain buffer, resuspended in 4 mL calcium stain buffer, and split into 4 tubes. Prior to calcium measurement, cells were incubated with biotinylated proteins/antibodies (1 μg/mL CD19-biotin (Accro Biosystems, CD9-H82E9) , 0.5 μg/mL anti-CD28-biotin (Biolegend,302904)) for 5 minutes at 37°. Baseline indo-1AM fluorescence was measured for 30 seconds, before addition of 20μg/mL avidin to crosslink biotinylated proteins. Calcium flux was measured for 5 minutes before addition of 1X cell stimulation cocktail (Invitrogen, 00–4970). Multiplexed populations were deconvoluted and calcium plots generated in FlowJo software (BD), and area under the curve measurements made using Prism software (GraphPad).

At 12–14 days in vitro (DIV) or 21 DIV, neuronal cultures were incubated with 6 µM indo-1/AM dye (Invitrogen) and 2 µM sulforhodamine 101 (SR101; Life Technologies) for 45 min at 37 °C. Images were acquired using a 25× water immersion objective on an inverted Zeiss LSM 510 multiphoton microscope with a humidified environmental chamber that maintained temperature at 37 °C. Two-photon excitation was performed at 750 nm using a mode-locked Chameleon Ti:sapphire laser (Coherent, Inc) as the source. Ratiometric images were generated with the simultaneous acquisition using non-descanned detectors of two spectral channels: 390–465 nm and 480–522 nm. Multiple fields were acquired that included 300–400 cells per dish. After baseline calcium was obtained, the cultures were treated with antibody-immunodepleted AβOs or AβOs-alone by replacing half of the total volume of the media in the dish with oligomer-containing media for 45 min. The cultures were then re-imaged in the same areas in the dish.