Journal: BMC Microbiology

Article Title: The non-pathogenic mycobacteria M. smegmatis and M. fortuitum induce rapid host cell apoptosis via a caspase-3 and TNF dependent pathway

doi: 10.1186/1471-2180-10-237

Figure Lengend Snippet: Differences in apoptosis induced by facultative-pathogenic versus non-pathogenic mycobacteria in primary murine macrophages . Differentiated BALB/c BMDMs were infected at an MOI of 10:1 with M. smegmatis (Msme), M. fortuitum (Mfort), M. kansasii (Mkan), M. bovis BCG or left untreated (UT). The percentage of apoptotic cells was determined using a propidium iodide based staining protocol to detect the population of hypodiploid cells via flow cytometry at 20 h after infection. Representative histograms are shown in A. B . The average and standard deviation of three independent experiments is shown. For this and all subsequent figures asterisks indicate statistically significance with * = 0.05>p > 0.01, ** = 0.01>p > 0.001 and *** = p < 0.001 which was determined by using one way ANOVA using GraphPad Prism5.0 software.

Article Snippet: Cells were collected after infection, pelleted and resuspended in propidium iodine (PI)/RNase buffer (BD Pharmingen) for 20 min and the percentage of hypodiploid positive cells was determined by flow cytometry in duplicates in the FL-2 channel at 580 nm (FACS-Calibur, BD Biosciences).

Techniques: Infection, Staining, Flow Cytometry, Standard Deviation, Software

Journal: BMC Microbiology

Article Title: The non-pathogenic mycobacteria M. smegmatis and M. fortuitum induce rapid host cell apoptosis via a caspase-3 and TNF dependent pathway

doi: 10.1186/1471-2180-10-237

Figure Lengend Snippet: PI-LAM of fast-growing mycobacteria induces apoptosis and IL-12 gene expression in macrophages . A . Differentiated human THP-1 cells were not treated (UT) or incubated with the indicated lipoglycans at 20 μg/ml for 24 h. The percentage of apoptotic cells was determined as Annexin-V-Alexa488-positive and propidium iodide-negative cells out of 10,000 analyzed cells by flow cytometry. B . The induction of Il-12 gene expression was analyzed by incubating a murine macrophage (RAW/pIL-12-GFP) reporter cell line which has the IL-12p40 promoter in front of the GFP gene, with the indicated lipoglycans for 16 h. GFP-expression was analyzed on 5,000 cells and the mean and standard deviation of three independent experiments is shown.

Article Snippet: Cells were collected after infection, pelleted and resuspended in propidium iodine (PI)/RNase buffer (BD Pharmingen) for 20 min and the percentage of hypodiploid positive cells was determined by flow cytometry in duplicates in the FL-2 channel at 580 nm (FACS-Calibur, BD Biosciences).

Techniques: Expressing, Incubation, Flow Cytometry, Standard Deviation

Journal: BMC Microbiology

Article Title: The non-pathogenic mycobacteria M. smegmatis and M. fortuitum induce rapid host cell apoptosis via a caspase-3 and TNF dependent pathway

doi: 10.1186/1471-2180-10-237

Figure Lengend Snippet: Mycobacteria do not induce rapid apoptosis in BMDM originating from C57Bl/6 mice . A . Differentiated C57Bl/6 BMDMs were infected at an MOI of 10:1 with M. smegmatis (Msme), M. fortuitum (Mfort), M. kansasii (Mkan), M. bovis BCG or left untreated (UT). The percentage of apoptotic cells was determined using a propidium iodide based staining protocol to detect the population of hypodiploid cells via flow cytometry at 20 h after infection. B . C57Bl/6 BMDMs were infected as in A. or incubated with staurosporine (ST) and the amount of apoptosis was detected using TUNEL staining and flow cytometry analysis. C . Macrophages were infected at MOIs of 1:1, 3:1, and 10:1 with M. smegmatis (Msme), M. fortuitum (Mfort), M. kansasii (Mkan), M. bovis BCG, or left untreated (UT). Culture supernatants of triplicate wells were collected after 20 h and the amounts of secreted TNF was determined using ELISA. In A. and B. the data shown is the mean and standard deviation of three independent experiments. In C. the values are the mean and standard deviation of triplicate readings of one experiment and they are representative of three independent experiments.

Article Snippet: Cells were collected after infection, pelleted and resuspended in propidium iodine (PI)/RNase buffer (BD Pharmingen) for 20 min and the percentage of hypodiploid positive cells was determined by flow cytometry in duplicates in the FL-2 channel at 580 nm (FACS-Calibur, BD Biosciences).

Techniques: Infection, Staining, Flow Cytometry, Incubation, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation