Live dead fixable red stain

To detect mycobacterial escape from the phagosome, the CCF4 FRET assay was performed as described previously (21 (link)). Briefly, cells were stained with 8 μM CCF4 (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂–5 mM glucose, 25 mM HEPES [pH 7.3]) containing 2.5 μM probenecid (Sigma-Aldrich) for 1.5 h at room temperature. Live populations were distinguished from dead ones by addition of LIVE/DEAD fixable red stain (Invitrogen) for 30 min at room temperature. Following staining, cells were fixed with 4% paraformaldehyde (PFA) overnight and analyzed by flow cytometry (BD FACSCanto). Representative examples of the flow cytometry gating strategy to determine loss of FRET in infected cells can be found in Fig. S9c.

To detect mycobacterial escape from the phagosome, the CCF4 FRET assay was performed as described previously [34 (link)]. Briefly, cells were stained with 8μM of CCF4 (Invitrogen) in EM buffer (120mM NaCl, 7mM KCl, 1.8mM CaCl₂, 0.8mM MgCl₂ 5mM glucose, 25mM Hepes, pH7.3) containing 2.5μM of probenecid (Sigma-Aldrich) for 1.5 hours at room temperature. Live populations were distinguished from dead ones by addition of Live/Dead Fixable Red stain (Invitrogen) for 30 minutes at room temperature. After staining cells were fixed with 4% PFA overnight and analyzed by flow cytometry (BD FACS CantoII). 40,000 cells were acquired and post acquisition analysis done using FlowJo software (Treestar, OR). For estimation of bacterial β-lactamase activity, bacteria were resuspended in PBS containing 50μg/ml porcine esterase liver extract and 100nM CCF4-AM and incubated at 37°C for 12h. Fluoresence measurements were made using Biotek Synergy 4 microplate reader.

PBMC samples from both CLL patients and HC were enriched for NK cells by CD19 depletion, the percentage of NK cells was determined by flow cytometry, and cells were rested O/N in an incubator at 37°C. The next day, cells were co-cultured with K562 or Daudi cells (NK:target ratio of 1:10) or stimulated with PMA/Ionomycin, or left untreated for 4 hours at 37°C in the presence of brefeldin A (10 μg/ml, Sigma, St Louis, MO), GolgiStop and CD107a PE-Cy7 (BD Biosciences). Afterwards, cells were washed with PBA, and stained with CD56 BUV395, CD3 V500 (BD Biosciences) and Live/Dead Fixable Red Stain (Invitrogen) for 30 minutes at 4°C. Then cells were washed with PBA, fixed and permeabilized (Cytofix/Cytoperm reagent, BD Biosciences) and stained intracellularly for 30 minutes at 4°C with IFNγ BV421, pS6 (Ser240/244, Cell Signaling, Danvers, MA) and granzyme B AF700 (BD Biosciences). Cell were washed, resuspended in PBA, and analyzed on a LSR Fortessa flow cytometer (BD Biosciences). Data analysis was performed using Flowjo Mac Version 10. Gating strategy can be found in supplemental Figure 5B (Supplemental Digital Content).