Anti cd45.1 pe cy7

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
(λ_em 500–550 nm and λ_em 410–470
nm), APC (λ_em 660 nm)-, PerCp-Cy5.5 (λ_em 696 nm)-
or PE-Cy7 (λ_em 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).

Congenic Cd45.1 mice were injected i.p. with ID8A-luc cells (4 × 10⁶ cells per mouse). Th17–T_reg subsets (IL-17A⁺Foxp3^neg, IL-17A⁺Foxp3⁺, IL-17A^neg Foxp3^neg and IL-17A^negFoxp3⁺) were generated from Foxp3^GFP reporter mice as described above and the same number of cells was injected i.p. (0.5–2.0 × 10⁵ cells per mouse, n=4 mice per group) on days 5, 12 and 19. Control mice were left untreated. Mice were imaged using the IVIS platform (Perkin Elmer). On days 40±2, mice were killed to purify the cells from the spleens and the ascites. The cells were activated for 3 h with PMA (50 ng ml⁻¹) and ionomycin (0.75 μg ml⁻¹). A Mouse IL-17 Secretion Assay was used to detect IL-17-secreting cells. The cells were also stained for anti-CD4-APC (6 μg ml⁻¹, eBioscience, clone: GK1.5), anti-CD45.1-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: A20) and Fixable Viability Dye-efluor 780.