He means seM of 7 mice nies were counted 28 d after incubation at 37 . per group of a representative experiment. Flow cytometry. For flow cytometric analysis, the entire right lung was removed and processed as described for CFU determination. After incubation in digesMaterials and Methods tion solution, the organs were filtered, and the cell suspensions Plasmid DNA production and quality control. DNA-hsp65 was were treated with 1 ml of erythrocyte lysis solution (0.15 M prepared as described previously.5 Briefly, the hsp65 gene from NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA) for 1 min. The1100 Human Vaccines Immunotherapeutics Volume 9 Issue013 Landes Bioscience. Do not distribute.Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.AcknowledgmentsThis study was supported by the Funda o de Amparo Pesquisa do Estado de S Paulo, FAPESP (Grant number 2006/059638). We are thankful to Dr Patricia Dillenburg-Pilla and the National Institutes of Health Fellows Editorial Board (NIHFEB, Bethesda, USA) for a critical review of the manuscript.NoteAt the time this article was in press, the Food and Drug Administration (FDA) of the United States of America approved bedaquiline, the first new anti-tuberculosis drug in more than 40 y. Bedaquiline is approved only for MDR-TB cases and should be used in combination with other drugs.www.landesbioscienceHuman Vaccines Immunotherapeutics013 Landes Bioscience. Do not distribute.cells were washed, counted and checked for cell viability with Tripan blue 0.5 staining. The cells were plated at 1.0 106 cells per well and incubated in complete RPMI-1640 medium in the presence of 20 g/ml concanavalin A (Con-A) (SigmaAldrich) for 6 h. After incubation, the cells were washed and incubated at 4 for 30 min with FcBlock (supernatant of 2.4G2 cell culture, ATCC-HB-197). Cells were stained with surface markers and intracellular cytokines using the Cytofix/Cytoperm Plus Fixation/Permeabilization kit with BD Golgi Plug (BD Biosciences) following manufacter’s instructions. The following fluorochrome-labeled mAb were used: anti-TCR-FITC (clone GL3), anti-CD4-PerCP (clone RM4), anti-CD8-APC (clone 53-6.7), anti-IFN–PE (clone XMG1.2) anti-IL-17-PE (clone TC118H10). The isotype controls were: IgG1-PE (clone R3-34), IgG2-FITC (clone B81), IgG2a,-PerCP and IgG2a,-APC (clone R35-95). The cells were fixed in 2 paraformaldehyde overnight before removal from the level III bio-safety room. Samples were run in a FACSCantoTM I flow cytometer (BD Biosciences) using the FACSDiva 6.1.3 software for data acquisition. A minimum of 100,000 events was acquired per sample and analysis was performed on live, single cell lymphocytes using FlowJo software (Tree Star Inc.Honokiol ).Tarcocimab ELISPOT.PMID:24463635 ELISPOT assay was conducted using Mouse IFN- Set (BD Biosciences) according to the manufacturer’s instructions. Briefly, 100 l of anti-IFN- capture antibody solution (5 g/ml) were distributed to each well of the ELISPOT plate, following overnight incubation at 4 . Coating was discarded and blocking solution was added to avoid unspecific results. Once plates were prepared, 1.0 105 lung cells suspensions were incubated with 10 g/ml of recombinant Hsp65 in complete RPMI medium at 37 , 5 CO2 and 99 humidity for 48 h. Con-A was used as control. Cell suspensions were aspirated and wells were washed and filled with100 l of biotinylated detection antibody solution (2 g/ml) for 2 h at room temperature. The wells were washed and fille.
