Facsaria flow sorter

Highly purified populations of CD4⁺ or CD8⁺ T cells were isolated using a FACS-Aria flow-sorter (BD Biosciences). Total RNA was extracted using an RNeasy-Plus Mini-Kit (Qiagen, Hilden). Assessment of RNA quality, labelling and hybridisation to 8 × 60K Agilent Whole-Human-Genome-Oligo-Microarrays was performed according to published methods^{12 (link)}. Fluorescence signals from hybridised microarrays were detected using Agilent’s Microarray Scanner System (G2505C, Agilent-Technologies, Palo Alto). The Agilent Feature Extraction Software 10.7.3.1 was used to extract the microarray image files. Background-corrected intensity values were quantile-normalised and log₂-transformed. Only reporters with at least three valid signal intensity values in at least one sample group were considered. For visualisation in heatmap format, the log₂ intensity values were median-centred for each reporter. One-way analysis of variance (ANOVA) was performed using GeneSpring-GX v.11.5.1 (Agilent).

After PET imaging, mouse hearts and aortas were harvested and collected in PBS-filled tubes. Aortas were minced and digested with an enzymatic digestion solution containing liberase TH (4 U/ml) (Roche, Basel, Switzerland), DNase I (40 U/ml) and hyaluronidase (60 U/ml) in PBS. Heart tissue was minced and digested using an enzymatic digestion solution containing DNase I (60 U/ml), collagenase type I (450 U/ml), collagenase type XI (125 U/ml) and hyaluronidase (60 U/ml) in PBS. Samples were treated with the respective enzymatic solution for 60 min at 37 °C. All enzymes were purchased from Sigma-Aldrich (St. Louis, MO). Samples were then passed through a 70 μm filter, washed and prepared for antibody staining and flow sorting. Cellular fragments and debris were gated out of the analysis by utilizing forward and side angle light scatter signal. Macrophages were identified as Ly6G⁻ (Clone 1A8, PE/Cy7), CD11b⁺ (Clone M1/70, PE), CD11c⁻ (Clone N418, PerCP/Cy5.5) and F4/80⁺ (Alexa Fluor^® 647, Clone BM8) from Biolegend (San Diego, CA). The remaining CD11b⁺ cells were identified as Ly6G^lo, CD11b^hi, CD11c^hi. Data were acquired on a FACS Aria flow sorter (BD Biosciences, East Rutherford, NJ) and analyzed using FlowJo v10.0.7 (Tree Star, Ashland, OR). Sorted cells were gamma counted and activity per cell values were calculated.

D15 cells differentiated on beads according to cluster B protocols were harvested with 0.25% trypsin/EDTA (Life technologies) and diluted with staining/blocking solution (3% FBS in DPBS). Prior to staining with specific antibody or the corresponding isotype control, cells were incubated with mouse BD Fc block (#553142, BD Biosciences). The antibodies and corresponding isotype controls used were anti-mouse CD11b-APC (clone M1/70) and APC-Rat IgG2a isotype control, both purchased from BD Biosciences. Cells were incubated with antibody for 1 hour on ice, washed twice with 3% FBS in PBS, and then sorted using a FACS Aria flow sorter (BD Bioscience). The live cells were gated using a propidium iodide (PI) stained control and the sorting gates were set using the isotype control stained cells. Both the CD11b positive and the CD11b negative cells were collected. The CD11b positive cells were plated into 2 wells of a 96 well tissue culture plate in Stemline medium (Sigma) containing TPO, IL3 and IL6 (All from R&D) and incubated at 37°C for 1 h to allow the cells to attach. After the incubation period the media were removed and the cells subjected to the pHrodo assay (Life Technologies) as above, then counterstained with calcein green. Cells were subsequently photographed using a Nikon Eclipse 2000 epifluorescence microscope.

Thirteen days following initial activation, transduced T cells were sorted into pure populations of GD2-CAR⁺ αβ, Vδ1⁺, and Vδ2⁺ by FACS using a BD FACSAria flow sorter. ZOL-activated cells were stained with Vδ2 PE antibody and QBend10 APC. Vδ2⁺/QBend10⁺ cells were selected for further analysis. Transduced CD3/CD28 antibody-activated cells were stained with CD56 PE (BioLegend, clone 188) and QBend10 APC antibodies; CD56⁻/QBend10⁺ cells were selected for further analysis.
NTD control CD3/CD28 antibody-activated αβ cells were depleted of CD56⁺ natural killer (NK) cells by CD56 MicroBeads according to the manufacturer’s instructions (Miltenyi, 130-050-401). NTD ZOL-activated γδT cells were isolated using the TCR γ/δ+ T Cell Isolation Kit (Miltenyi, 130-092-892).
For ConA expansions, γδT cells were positively selected using TCR γ/δ MicroBeads (Miltenyi, 130-050-701) before FACS to reduce the numbers to be sorted. Pure populations of transduced (QBend10⁺) and NTD (QBend10⁻) αβ, Vδ1⁺, and Vδ2⁺ cells were obtained by staining with Vδ1 PE, Vδ2 FITC, anti-TCR γδ PE-Vio770, and QBend10 APC before sorting the respective cell populations.