Carboxyfluorescein diacetate succinimidyl ester

CD34⁺ CB cells (1 × 10⁶ cells/ml) were labeled with 5 μl carboxyfluorescein diacetate succinimidyl ester (Biolegend, Cat: 423801) according to the manufacture’s guidelines. Labeled cells were cultured for 24 h before they were stained with APC-labeled anti-human CD34 antibody (Biolegend, Cat: 343608) and sorted for CD34⁺CFSE⁺ cells using a FACS MoFlo (Beckman). Sorted cells were then resuspended in HSC expansion medium supplemented with DMSO (0.01%) or JNK-IN-8 (2 µM). Cell aliquots after 2 and 4 days in culture were stained with APC anti-human CD34 (Biolegend, Cat: 343608) and APC-Cy7 anti-human CD45RA antibody (Biolegend, Cat: 304128) before cells were analyzed for CFSE intensity by FCS express version 6 software.

Autologous CD4⁺ T-lymphocytes were obtained from the same healthy PPD⁺ donors according to the isolation protocol described above. Positive selection of the CD4⁺ lymphocytes was performed using anti-CD4 magnetic beads (Miltenyi Biotec). Isolated lymphocytes were cultivated in 75 cm² flask at 2 × 10⁶ cells per mL in RPMI-1640 medium (HyClone, GE Healthcare) supplemented with 15% (v/v) FBS (HyClone, GE Heaclthcare), 1 mM sodium pyruvate (HyClone, GE Heaclthcare), 10 mM HEPES (HyClone, GE Heaclthcare) and 20 UI/ml of human recombinant Interleukin 2 (Biolegend) for 3 days prior to the experiment. Immediately before the experiment the lymphocytes were stained with Carboxyfluorescein diacetate succinimidyl ester (Cat # 423801, Biolegend) following the manufacturer’s instructions. Macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and treated for 24 h were washed and cocultivated with the lymphocytes at a ratio of 5 lymphocytes per macrophage for 5 days. CD4⁺ lymphocytes were recovered after 5 days of coculture and analyzed using Guava easyCyte™ 5HT flow cytometer.