Telotaggg telomere length assay

Telomere length in each of the tissues was assessed using TeloTAGGG^™ Telomere Length Assay (Sigma-Aldrich, St Louis, MO) as per manufacturer’s recommendation and described elsewhere (Tarry-Adkins and Ozanne 2018 (link)). Briefly, about 1.0 μg DNA was digested with HinfI and RsaI restriction enzymes for 2 h at 37 °C and the restriction enzyme reaction quenched by adding 5× SDS loading buffer (Roche Diagnostics, Mannheim, Germany). The samples were than loaded onto agarose gels containing ethyl bromide (Sigma-Aldrich) and separated by pulsed field gel electrophoresis. The undigested and digested DNA were checked for non-specific degradation and digestion by the restriction enzymes by visualizing under UV light (BioRad). The separated DNA fragments were transferred to nylon membrane (Roche Diagnostics) by Southern blotting, and dioxygenin labelled telomeric repeat length was determined with anti- dioxygenin antibodies using chemiluminescent detection. The densitometry of the telomere signals was analyzed using ImageJ (National Institutes of Health, Bethesda, MD) as described previously (Tarry-Adkins and Ozanne 2018 (link)).

Autologous APCs and primary human T cells (5x10⁶) were conjugated 1:1 with for 24h in the presence of antigen pool described above. The day after, conjugates were broken using a 2-ml syringe with cold PBS plus 5mM EDTA and T cell and APC fractions purified by CD3 Microbeads described above. In parallel, identical numbers of T cells and APCs were left unconjugated for comparison of telomere length before and after forming the synapse. Total genomic DNA was used as loading control and detected by anti-dsDNA antibodies (1:10000; Abcam ab27156). Genomic DNA extracted using the PureLink™ Genomic DNA Mini Kit (K182001, ThermoScientific). Telomere length was analysed following the TeloTAGGG™ Telomere Length Assay (12209136001, Sigma-Aldrich).
For TRF on APC vesicle preparations, 1.5 μg of vesicle DNA (recovered after final 100,000g ultra-centrifugation of APC supernatants) were analysed in the presence or in the absence of HinF I/Rsa I (10729124001 Sigma-Aldrich; ER0801 Thermo-Fisher) restriction enzymes to digest non-telomeric DNA (1,5U, 60 min at 37°C).