Experimental infection  Mycobacterium avium strain 2447 (smooth t

Experimental infection. Mycobacterium avium strain 2447 (smooth transparent variant kindly provided by Dr F. Portaels from the Institute of Tropical Medicine, Antwerp, Belgium) was grown in Middlebrook 7H9 medium containing 0.05% Tween 80 at 37 °C until mid-log phase of growth. Bacteria were harvested by centrifugation and resuspended in saline containing 0.05% Tween 80. The bacterial suspensions were briefly sonicated with a Branson

sonifier to disrupt bacterial clumps, diluted, and stored in aliquots at −70 °C until use. Intravenous infection with M. avium was performed through the tail lateral vein with 106 CFU per animal. At specific time-points (4, 8 and 20 weeks post infection), the organ bacterial load was determined as previously described [21]. Briefly, mice were anaesthetized and killed with isoflurane (Abbott, IL, USA). The organs were removed in aseptic conditions, homogenized, and serial dilutions were click here prepared in distilled sterile water with 0.05% Tween 80 and plated onto Middlebrook 7H10 agar medium. The numbers of CFU were

counted after 1 week of incubation at 37 °C. Flow cytometry.  Single cell suspensions were prepared from the spleen and the thymus of each mouse. Spleen erythrocytes were lysed with a haemolytic solution (155 mm NH4Cl, 10 mm KHCO3, Sunitinib nmr pH 7.2). For each staining, 5 × 105 cells from each organ were incubated with a specific set of antibodies for 20 min at 4 °C. Cell surface markers were analysed using anti-CD25 APC or Pe (clone PC61), anti-CD11b PE (clone M1/70), anti-CD3 FITC, PE or APC (clone 145-2C11), anti-CD4 FITC or PECy5 (clone RM4-5), anti-CD62L FITC (clone MEL-14), anti-CD44 PE (clone IM7), anti-CD19 FITC (clone 6D5), anti-NK-1.1 FITC (clone PK136), anti-CD8 FITC, APC or APCCy7 (clone 53-6.7) and anti-Ly-6G/Ly-6C PECy5 (Gr-1;

clone RB6-8C5) (all from Biolegend, San Diego, CA, USA). Cells were Niclosamide fixed with 2% formaldehyde after staining. The analysis of the cell populations was based on the acquisition of 30,000 events using CellQuest software on a FACscalibur flow cytometer or a FACSAria cell sorter (Becton Dickinson, NJ, USA). Data analysis was performed using FlowJo software (Tree Star, Inc, Ashland, OR, USA). Detection of IFN-γ in serum samples.  Mice were anaesthetized with isoflurane (Abbott, IL, USA), and retro-orbital bleeding was performed before killing. Blood was allowed to clot and serum was collected after centrifugation and frozen at −80 °C until use. Quantification of IFN-γ was done by a two-side sandwich ELISA using anti-IFN-γ-specific affinity-purified mAbs (R4-6A2 as capture and biotinylated AN-18 as detecting mAbs), and the standard curves were generated with known amounts of IFN-γ (Peprotech, Rocky Hill, NJ, USA). The sensitivity of the assay was 20 pg/ml. Statistical analysis.  All data are presented as means + SD.

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