F10 medium

Detection of TCR-specific unscheduled DNA synthesis was performed essentially as previously described^{39 (link)}. Primary XP168LV (XP-C patient cells) were transfected with siRNAs and subsequently serum starved for at least 24 h in F10 medium (Lonza) supplemented with 0.5% FCS and antibiotics. Cells with subsequently irradiated with 8 J/m² UV-C, and pulse-labeled with 20 µM 5-ethynyl deoxy-uridine (EdU; Invitrogen) and 1 µM FuDR (Sigma–Aldrich) for 8 h. After labeling, cells were chased with F10 medium supplemented with 0.5% FCS and 10 µM thymidine for 15 min, and fixed for 15 min with 3.6% formaldehyde and 0.5% Triton-X100 in PBS. Next, cells were permeabilized for 20 min in PBS with 0.5% Triton-X100 and washed and stored in 3% bovine serum albumin (BSA, Thermo Fisher) in PBS. The incorporated EdU was visualized by click-it chemistry-mediated binding of Biotin (Azide-PEG3-Biotin Conjugate; Jena Biosciences) using the protocol and reagents from the Invitrogen Click-iT EdU Cell Proliferation Kit for Imaging (Invitrogen), and signals were amplified using protocol and reagent of the Alexa Fluor-488 Tyramide streptavidin SuperBoos Kit (Thermo Fisher). After click-it and amplification, cells were counterstained with 0.1 μg/mL DAPI, washed extensively with 0.1% Triton-X100 in PBS and mounted in Polymount (Brunschwig).

Preparation of limb muscle for flow cytometric analysis was adapted from Liu et al. [28 (link)]. In short, dissected tissue was minced thoroughly to small pieces in F10 medium (Lonza) containing collagenase II (750 U/ml; Fisher Scientific) using scalpels. Minced tissue was dissociated for 70 min in F10 medium containing 750 U/ml collagenase II), then for 30 min in F10 medium containing collagenase II (100 U/ml) and dispase (1.1 U/ml; Fisher Scientific). Cell suspensions were filtered over a 40 μM cell strainer (Falcon) and a small sample was retrieved to determine total mononuclear cell count using a hematocytometer. The cell suspensions were stained with CD31-APC (1:100), CD45-APC (1:100), Sca1-FITC (1:100) and Vcam-biotin (1:50) primary antibodies. Vcam was visualized using streptavidin-PECY7 (1:100). All antibodies for flowcytometry were derived from BD Biosciences. Cell viability was determined by staining with 1 μg/ml Hoechst 33258 (Sigma). Samples were analyzed on a BD-ARIAIII (BD biosciences).