The principle of the β-lactamase CCF-4 FRET assay is summarized in S1 Fig. . To
measure the Mtb phagosomal rupture, cells were stained during 1h at
RT, with 8 μM CCF-4 (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM Hepes, pH 7.3)
complemented with 2.5 μM probenecid. Cells were then stained with
anti-CD11c-PE-Cy7, anti-CD11b-PerCp-Cy5.5 (eBiosciences) or anti-CD11b-APC (BD) mAbs
andfixed with 4% PFA overnight at 4°C. Cell mortality in the same cultures of
infected cells was determined by use of Pacific Blue Dead/Live reagent (Invitrogen),
which reacts with free amines both inside and outside of the plasma membrane,
yielding log10 1 more intense fluorescent staining of dead cells.
Anti-CD45.1-PE-Cy7 and anti-CD45.2-PerCpCy5.5 were from eBiosciences. To avoid
fluorochromes with emission signals overlapping with those of CCF-4
(λem 500–550 nm and λem 410–470
nm), APC (λem 660 nm)-, PerCp-Cy5.5 (λem 696 nm)-
or PE-Cy7 (λem 778 nm)-conjugated mAbs were chosen for concomitant
cell surface staining. Cells were analyzed in a CyAn cytometer using Summit software
(Beckman Coulter, France). At least 100,000 events per sample were acquired for
in vitro assays. For in vivo detection of CCF-4
signal in CD45 congenic mouse model, 1,000,000 events per sample have been acquired.
Data were analyzed with FlowJo software (Treestar, OR).
measure the Mtb phagosomal rupture, cells were stained during 1h at
RT, with 8 μM CCF-4 (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM Hepes, pH 7.3)
complemented with 2.5 μM probenecid. Cells were then stained with
anti-CD11c-PE-Cy7, anti-CD11b-PerCp-Cy5.5 (eBiosciences) or anti-CD11b-APC (BD) mAbs
andfixed with 4% PFA overnight at 4°C. Cell mortality in the same cultures of
infected cells was determined by use of Pacific Blue Dead/Live reagent (Invitrogen),
which reacts with free amines both inside and outside of the plasma membrane,
yielding log10 1 more intense fluorescent staining of dead cells.
Anti-CD45.1-PE-Cy7 and anti-CD45.2-PerCpCy5.5 were from eBiosciences. To avoid
fluorochromes with emission signals overlapping with those of CCF-4
(λem 500–550 nm and λem 410–470
nm), APC (λem 660 nm)-, PerCp-Cy5.5 (λem 696 nm)-
or PE-Cy7 (λem 778 nm)-conjugated mAbs were chosen for concomitant
cell surface staining. Cells were analyzed in a CyAn cytometer using Summit software
(Beckman Coulter, France). At least 100,000 events per sample were acquired for
in vitro assays. For in vivo detection of CCF-4
signal in CD45 congenic mouse model, 1,000,000 events per sample have been acquired.
Data were analyzed with FlowJo software (Treestar, OR).