far red fluorescent polystyrene beads bangs fsfr002  (Bangs Laboratories)

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense

doi: 10.1371/journal.ppat.1009943

Figure Lengend Snippet: (A) Bacterial burden expressed as colony forming units (CFU) after 24h of S. Typhimurium infection in 2-DG-treated WT BMDMs compared to untreated (UT) controls (n = 5). (B) Intracellular S. Typhimurium load in WT and IR Δmyel BMDMs after 24h of infection (n = 3). (C) S. Typhimurium in 4-OHT-treated WT BMDMs compared to untreated (UT) controls after 24h of infection (n = 3). (D) L. monocytogenes (E) S. aureus burden in 2-DG-treated WT BMDMs compared to untreated (UT) controls (n = 3). (F) S. aureus burden in WT and IR Δmyel BMDMs (n = 3) 24h post-infection. (G) Bacterial load in livers of WT and IR Δmyel mice after 3 days post S . Typhimurium infection or (H) 4-OHT-treated mice. Data represent 2 experiments with 5 mice each. Data are shown as mean ± S.E.M. and statistical significance calculated using student t-test and represented as * = p<0.05; ** = p<0.01; *** = p<0.001. (I) MFI of OVA323-339-MHC II complexes on the surface of WT BMDMs pre-treated with 2-DG and LPS (n = 3). (J) MFI of unprocessed Alexa647-labelled OVA in phagosomes isolated from 2-DG-treated BMDMs analyzed by flow cytometry (n = 3). All samples were pre-stimulated with LPS. (K) MFI of unprocessed Alexa647-labelled OVA in bead containing phagosomes isolated from S. Typhimurium-infected BMDMs analyzed by flow cytometry (n = 3).

Article Snippet: To assess the β-galactosidase activity in phagolysosomes, red fluorescent beads (Bangs Laboratories) were coated with 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C 12 FDG, Life Technologies) for 60 min at 37°C in NaHCO 3 pH 9.6 buffer.

Techniques: Infection, Isolation, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense

doi: 10.1371/journal.ppat.1009943

Figure Lengend Snippet: (A) MFI of pH-sensitive pHrodo-E. coli particles in BMDMs untreated (UT) or pre-treated with 2-DG, normalized to Alexa647 MFI (n = 3). (B) MFI of pHrodo-E. coli particles in WT and IR Δmyel BMDMs normalized to Alexa647 MFI (n = 3). (C) MFI of pHrodo-E. coli particles in S. Typhimurium-infected WT BMDMs (2h p.i.) normalized to Alexa647 MFI (n = 3). (D) MFI of pHrodo-E. coli particles in 4-OHT-treated BMDMs infected with S. Typhimurium for 2h normalized to Alexa647 MFI (n = 3). (E-F) Expression of v-ATPase subunits (V0, V1) in isolated bead-containing phagosomes from untreated and 2-DG-treated BMDMs. The image shown is representative of 3 individual experiments. Immunoblot band intensities were quantified using ImageJ and the V1/ V0 ratio was determined and plotted (n = 3). (G-H) Expression of v-ATPase subunits (V0, V1) in isolated bead phagosomes from WT and IR Δmyel BMDMs. Western blot was quantified and V1/V0 ratios are shown. Expression of v-ATPase subunits (V0, V1) in isolated S. Typhimurium phagosomes and cytoplasm. V1/V0 ratios were quantified and plotted. (I-J) V0 subunit A was immunoblotted from isolated S . Typhimurium phagosomes and probed for V1 subunit B. (K-L) Proximity Ligation Assay (PLA) analysis of V0-aldolase A interaction in 2-DG treated BMDMs. The image shown is representative of 3 individual experiments. (M) Immunoprecipitation of V0 subunit from S . Typhimurium infected phagosomes at indicated times probed for V1, V0 and actin. Rabbit IgG was used as a control for immunoprecipitation. (N) Phagosomes isolated from 2DG-treated and untreated macrophages were subjected to Native-PAGE and immunoblotted for v-ATPase subunits V0 and V1.

Article Snippet: To assess the β-galactosidase activity in phagolysosomes, red fluorescent beads (Bangs Laboratories) were coated with 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C 12 FDG, Life Technologies) for 60 min at 37°C in NaHCO 3 pH 9.6 buffer.

Techniques: Infection, Expressing, Isolation, Western Blot, Proximity Ligation Assay, Immunoprecipitation, Control, Clear Native PAGE

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense

doi: 10.1371/journal.ppat.1009943

Figure Lengend Snippet: Schematic representation of S. Typhimurium-mediated evasion of phagosome degradation in macrophages by preventing glycolysis-regulated assembly of the v-ATPase.

Article Snippet: To assess the β-galactosidase activity in phagolysosomes, red fluorescent beads (Bangs Laboratories) were coated with 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C 12 FDG, Life Technologies) for 60 min at 37°C in NaHCO 3 pH 9.6 buffer.

Techniques: