7 aminoactinomycin d 7 aad

For EdU FACS, cells were treated with 10 μM EdU for 10 min, trypsinized, washed with PBS, and fixed with cold 70% ethanol on ice for 30 min to overnight. Cells were washed with PBS, and EdU staining was performed by using the EdU Click-iT kit (Thermofisher, # C10632), according to the manufacturer’s instructions. For DNA staining, we used 7-AAD (7-Aminoactinomycin D) (Thermofisher, # A1310) or FxCycle™ PI/RNase Staining Solution (Thermofisher, #F10797).
Chromatin association of MCM was assessed essentially as described^{46 (link)}: after trypsinization and PBS wash, cells were extracted with CSK buffer (10 mM PIPES pH 7.0, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl₂, 0.5% Triton X-100, protease inhibitors (Pierce #A32953), fixed with 4% paraformaldehyde, and blocked with 5% BSA followed by immunostaining with anti-MCM7 antibody (Santa Cruz, #sc-9966) at 1:200 dilution. 7-AAD (7-Aminoactinomycin D) (Thermofisher, # A1310) was used for DNA staining.
Flow cytometry was performed on an FACSCalibur flow cytometer, and data were analyzed by using FCSalyzer software. GraphPad Prism 9 was used for statistical analyses.

To generate IA^b/Myhc-α 334–352 dextramers, IA^b/CLIP precursors were prepared as described above. After biotinylation, IA^b molecules were treated with thrombin and CLIP was released. Myhc-α 334–352 was then loaded onto empty IA^b molecules by peptide-exchange reactions [33 (link)]. The dextramers were generated by incubating Myhc-α 334–352-exchanged IA^b molecules with fluorochrome-labeled dextran backbone [33 (link),45 (link)]. We used IA^s/Theiler’s murine encephalomyelitis (TEMV) 70–86 dextramers as controls [37 (link),39 (link)]. Lymphocytes obtained from mice immunized with Myhc-α 334–352 were stimulated with Myhc-α 334–352 (50 µg/mL) for two days and maintained in growth medium containing interleukin (IL)-2 in order to determine the frequency of antigen-specific T cells. Viable lymphoblasts were harvested by Ficoll–Hypaque density gradient centrifugation on day 5 (MP Biomedicals, Santa Ana, CA, USA), and the cells were kept in growth medium containing IL-2. Dextramer staining was carried out on day 9 using IA^b/dextramers, anti-CD4, and 7-aminoactinomycin D (7-AAD; Invitrogen, Carlsbad, CA, USA), as previously described [39 (link)]. After cells were acquired by flow cytometry (FACSCalibur™, BD Biosciences, San Jose, CA, USA), percentages of dextramer⁺ cells were determined in the 7-AAD⁻ (live) CD4⁺ subset using FlowJo software v10.9 (Tree Star, Ashland, OR, USA).

Splenocytes were stimulated with 100 U/ml of mIL-2, 10 µg/ml mIL-4, 1 µg/ml mIL-5 (all Peprotech) and 2 µg/ml F(ab′)2 fragments goat anti-mouse IgM (Jackson ImmunoREsearch), 2 µg/ml hamster anti-mouse CD40 mAb (3/23, BD), or 100 nM ODN74 5′–AAAAAAAAAAAAAACGTTAAAAAAAAAAA–3′ (Microsynth). Proliferation was assessed using the cell proliferation dye CPD eFluor 670 (eBioscience) and viability by 7-amino-actinomycin D (7AAD) or TO-PRO3 (Invitrogen) and Annexin-V exclusion and flow cytometric analysis.