FITC-Labeling of M. bovis BCG for Phagocytosis

M. bovis BCG (Tokyo 172 strain, Japan BCG laboratory #876391) was labeled with FITC (SIGMA #F7250), as described previously^{99 (link)}. Briefly, 6.0 × 10⁷ CFU of M. bovis BCG were resolved in 200 μl of 0.1 M carbonate–bicarbonate buffer (pH 9.6)/0.05% Tween 80 containing of 30 μg/ml FITC, and incubated at 37 °C for 15 min. The FITC-labeled mycobacteria were washed with PBS/0.05% Tween 80. For phagocytosis assay, WT or Trem2^−/− BMDMs (1 × 10⁵) were incubated with 1 × 10⁶ (MOI 10:1) of FITC-labeled mycobacteria in a 96-well plate for 4 h at 37 °C. Negative controls were prepared in identical conditions but incubated on ice (0 °C). The phagocytosis was stopped by washing with ice-cold PBS. The phagocytosed mycobacteria were analyzed by flow cytometer after addition of 0.02% Trypan Blue to quench FITC fluorescence of extracellular but not internalized mycobacteria¹⁰⁰ .