*2.7. Immunization of Mice and Isolation of Splenocytes*

Groups of eight adult (seven-week-old) female BALB/c mice were immunized intradermally in one footpad, and two groups were left unimmunized. The first group received 10<sup>5</sup> colony-forming units (CFU) of BCG.HTI2auxo.int (Group A), the second group received 10<sup>6</sup> CFU of BCG wt (Group B), both groups in one footpad. Two groups were left unimmunized (Groups C and D). ChAdOx1.HTI was constructed as previously described [36], and groups A–C were boosted intramuscularly with

10<sup>9</sup> viral particles (vp) after five weeks, while group D was left unimmunized. All the mice were sacrificed two weeks after the boost for immunogenicity analyses. Immediately following sacrifice of the animals, splenocytes were harvested and homogenized using 70 μm cell strainers (Falcon; Becton Dickinson, Franklin Lakes, NJ, United States) and 5-ml syringe rubber plungers. Red blood cells were removed with ACK lysing buffer (Lonza, Barcelona, Spain), and the splenocytes were washed and resuspended in complete medium (R10 (RPMI 1640 supplemented with 10% fetal calf serum and penicillin–streptomycin), 20 mmol/L of HEPES, and 15 mmol/L of 2-mercaptoethanol).
