Inside stain kit

2 × 10⁵ cells were fixed in ice-cold 70% ethanol while being vortexed, incubated on ice for 15 min, washed in PBS and resuspended in 250 μl of staining solution containing 20 ng/ml propidium iodide, 200 ng/ml RNAse A (Sigma-Aldrich), 0.1% Triton-X 100 and 1 μl CD19-FITC mAb in PBS. Cells were incubated for 15 min and submitted to flow cytometry. For concurrent cell cycle and apoptosis staining, 5 × 10⁶ cells were fixed and permeabilized using the Inside Stain Kit (Miltenyi Biotec) according to the manufacturer's instructions. Staining solution contained 20 ng/ml propidium iodide, 200 ng/ml RNAse A, 1 μl anti-cleaved caspase-3 antibody (Cell Signaling) and 1 μl DyLight-488 goat anti-mouse secondary antibody (Thermo Scientific). Cell cycle analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).

After 6 days of co-culture, CD4⁺ and CD8⁺ T cells were restimulated with 200 nM PMA (Sigma-Aldrich) plus 1 μg/ml of ionomycin (Sigma) for 4.5 hours. Brefeldin A (5 μg/ml, Sigma) was added during the last 2 hours. For intracellular cytokine production, cells were fixed and permeabilized with Inside Stain kit (Miltenyi Biotec) and stained with FITC-conjugated anti-IFN-γ (clone 45-15, Miltenyi Biotec), PE-conjugated anti-IL-4 (clone 7A3-3, Miltenyi Biotec), APC-conjugated anti-IL-13 (clone OES10-5A2, Biolegend), APC-conjugated anti GrB (clone REA226) following the manufacturer’s recommendations. Response definition criteria were defined post-hoc. Raw data can be provided per request.

For immunofluorescence staining the Inside Stain Kit (Miltenyi Biotech Ltd., Bisley, Surrey, UK; Miltenyi Biotech Inc., Auburn, CA, USA) was used with minor modifications. After the fixation step, the cover glasses were incubated with the anti-AHR primary antibody (1:100, Santa Cruz Biotechnology) overnight at 4 °C. The slides were then washed and incubated with the secondary antibody for 30 min at room temperature (1:500, anti-rabbit FITC, Bethyl Laboratories, Inc., Montgomery, TX, USA). The slides were mounted in the VECTASHIELD HardSet Mounting Medium with DAPI (VECTOR LABORATORIES, INC., Burlingame, CA, USA), and then analysed using a confocal microscope (see below).

The hiPSC-CMs were plated on glass coverslips at 0.7–1.3 × 10⁴/ cm² and fixed 6 days later using the Inside Stain Kit (Miltenyi) according to manufacturer's instructions. Fixed cells were incubated with α-actinin (1:250; Sigma; Cat# A7811) and myosin heavy chain (1:50; Miltenyi; Cat# 130-112-757) antibodies. The primary antibodies were detected with AF594- (1:200; Life Technologies; Cat# A-21203) and Vio515- (1:100; Miltenyi; Cat# 130-112-760) conjugated secondary antibodies, respectively. All antibodies were diluted in permeabilisation medium and incubated for 10 min. Cells were stained with 4′,6 Diamidino-2-Phenylindole (DAPI) (0.3 μM) for 5 min. Images were captured using a confocal laser scanning microscope SP8 (Leica).

96 h after Jurkat cell transduction with LeGO-CCR5-iB2-Puro+ or LeGO-(CCR5^Δ55-60)-iB2-Puro+, cells were harvested, washed with PBS, and resuspended in 250 µl PBS. Cells were fixed and permeabilised with the Inside Stain Kit (Miltenyi Biotec). Staining of cells was performed using 5 µl of the PerCP/Cy5.5 anti-human CD195 (BioLegend, San Diego, CA) and 5 µl of APC anti-human CD3 (Miltenyi) antibodies. After 15 min incubation in the dark at room temperature, cells were washed with PBS and resuspended in 100 µl fresh PBS. Cell images were obtained using the ImageStreamX Mk II System (Amnis/Luminex, Austin, TX); data were acquired and analysed with IDEAS Software package (Amnis/Luminex) using channels 5, 7, 11 and brightfield. Compensation was performed according to the software introduction using single-stained cells. BFP- (CCR5, or CCR5^Δ55-60) positive cells were gated and their images investigated. Normal erode masque was applied to all images, and the internalization wizard was used to check the relative BFP to Cy5.5 signal localisation (Internalisation of BFP signal by Cy5.5 signal marking CCR5). Cells with internalised BFP signals were selected by choosing the cell population with an internalisation score ≥1.