Canto 2 flow cytometer

For intracellular staining, 2 x 10⁶ PBMNCs were re-suspended in 1ml of complete medium and cultured in 15ml, 120x17mm, polypropylene tubes (SARSTEDT, Nümbrecht, Germany), in the absence (control) or in the presence of either TT (10μg/ml), PPD (15μg/ml) or C. Alb (10μg/ml). Anti-CD28 (clone CD28.2, eBioscience) was added to all tubes (1μg/ml). After 90 minutes, brefaldin A (Golgi Plug, BD Biosciences) was added (1/1000 dilution). After 6h, cells were washed and stained for surface markers first, and then they were permeabilized and stained for intracellular markers. Data were acquired on a Canto II flow cytometer and analyzed using FlowJo (7.6.5) (Treestar) software. For analysis of antigen-specific CD4⁺ T cells, between 1.5–1.8 x 10⁶ events in the singlet gate were acquired. Responses were considered positive if the frequency of the events in antigen-stimulated cultures was ≥ 0.01%, and the frequency of background events was ≤ 30% of the frequency of events in the antigen-stimulated cultures.

The myoblasts were harvested and fixed in PBS+1% PFA. They were then either (i) 0.5% saponin-permeabilized and stained with mouse monoclonal anti-KCa1.1 α antibodies (NeuroMab, Davis, CA, USA) to detect channel levels as described^{42 (link)} or (ii) 0.1% Triton X-100 permeabilized and stained with 7-amino actinomycin D (7-AAD) to label DNA for cell cycle analysis. In addition, 5-bromo-2′-deoxyuridine (BrdU) was used to label cells in culture before 7-AAD staining following the manufacturer's protocol. Fluorophore-conjugated secondary antibodies were used where appropriate (Invitrogen, Grand Island, NY, USA). The data were acquired with a Becton Dickinson Canto II flow cytometer and analyzed with FlowJo Version 7 (Treestar, Ashland, OR, USA). The Dean-Jett model was applied to evaluate G1/S/G2-phase DNA content (%).^{62 (link)} Analysis of cell cycle was performed with asynchronous population of cells because serum-starvation led to the induction of cell fusion into multinucleated cells, thereby directly affecting their cell cycle.