Bcg danish

M. bovis BCG-Danish was obtained from Statens Serum Institute (Copenhagen, Denmark) and was grown in Middlebrook 7H9 medium (Difco Laboratories, BD Diagnostic Systems, Sparks, MD) with oleic acid–albumin–dextrose–catalase (OADC Enrichment; Difco Laboratories, BD Diagnostic Systems) and 0.05% tyloxapol (Sigma-Aldrich, St. Louis, MO) (13 (link)). Bacteria were grown from low passage number frozen stocks, cultured to midlog phase, and then frozen in medium with 5% glycerol at −80°C. Bacteria were thawed, washed, resuspended in PBS containing 0.05% Tween-80, and sonicated to obtain a single-cell suspension prior to infection. Mice were vaccinated with 2 × 10⁶ CFU of bacteria s.c. at the base of the tail or i.v. in the tail vein. Mice were euthanized 4 wk after vaccination to isolate spleen and draining lymph nodes. Splenocyte and lymph node single-cell suspensions were prepared by gently forcing spleen through a 70-μm cell strainer. RBC lysis step was performed with splenocyte suspension using RBC lysing buffer (Hybri-Max; Sigma-Aldrich).

M. bovis Bacillus Calmette-Guérin (BCG)-Danish was obtained from Statens Serum Institute (Copenhagen, Denmark), and was grown in Middlebrook 7H9 medium (Difco Laboratories, BD Diagnostic Systems, Sparks, MD) with oleic acid-albumin-dextrose-catalase (OADC Enrichment; Difco Laboratories, BD Diagnostic Systems, Sparks, MD) and 0.05% tyloxapol (Sigma-Aldrich, St. Louis, MO). Mycobacterium smegmatis (strain mc²155) were grown in liquid cultures in Sauton medium [17 (link)]. Bacteria were grown from low passage number frozen stocks, cultured to mid-log phase and then frozen in medium with 5% glycerol at −80°C. Bacteria were thawed, washed, resuspended in PBS containing 0.05% Tween-80, and sonicated to obtain a single-cell suspension prior to infection. Mice were vaccinated with 2 × 10⁶ CFU of bacteria at the base of the tail unless otherwise indicated. Mice were euthanized 4–6 weeks after vaccination to isolate spleen and splenocyte suspensions were prepared by gently forcing spleen through a 70-μm cell strainer. RBC lysis was performed using RBC lysing buffer Hybri-Max (Sigma).