Cells were then washed in PBS and re-fixed in 4% formaldehyde. Some samples were thereafter

stained with 5 μg/mL FM4-64× (Molecular probes) in PBS for 30 s on ice and then fixed again with 4% formaldehyde without prior wash, in order to visualize the membrane of the cells during microscopy. Primary antibodies were from BD Biosciences. After staining, coverslips were mounted on a microscope slide, sealed with nail polish and stored dark at 4°C before imaging by confocal microscopy within 24 h. The slides were examined with an LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) equipped with a 63× objective, and using PF-562271 chemical structure the LSM software v. 3.2 (Carl Zeiss). Several representative images from each sample were acquired with similar scanning parameters (63× plan-apochromat/1.4 oil, confocal slide of 1–2 μm). Image analysis and quantification of co-localization was performed using the LSM software v. 3.2, and co-localization between vaccines with each other or with Lamp-1 was defined as overlapping fluorescence, and is shown with arrows on the representative images. Statistical differences between selected means were analyzed with a Student’s t-test. Whenever more than one comparison was made in the same experiment, the approximate Bonferroni correction was used, where samples are considered significant at the overall level of α if α (sample)<α (overall)/n, where n is the number

of comparisons. selleck chemical For multiple comparisons of more than three means, one-way ANOVA was used with Turkey’s post test for multiple comparisons, and statistical are differences marked by asterisks in figures and explained in figure legends. Statistics were performed

with comparisons of means from the one experiment shown in the figures only, and never between repeated experiments, since these were not completely matched regarding sample size and day of analysis. This work was supported by The Bill and Melinda Gates Foundation, the Tuberculosis Vaccine Cluster-European Commission selleck inhibitor (TBWA-EC) Grant contract no. CT2003–503367 and the Option Foundation. The authors thank Timothy Mark Doherty for critical reading and comments on the manuscript. The excellent technical assistance provided by Charlotte Fjordager, Kristine Persson, Benjamin Anderschou Holbech Jensen, Lene Rasmussen, Merethe Henriksen, Katja Bøgebjerg Carlsen and Janne Frandsen as well as the animal technicians at the Statens Serum Institut is gratefully acknowledged. Conflict of interest: P.A, C.A., J.D. and C.V. are co-inventors of patents relating to tuberculosis fusion protein vaccine Ag85B-TB10.4. All rights have been assigned to the Statens Serum Institut. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the article apart from those disclosed. “
“Citation Lee HJ, Kim H, Ku S-Y, Kim SH, Kim JG.