![]() The results were analysed with FlowJo software version 7.6.5 (Tree Star). Fifty thousand MDM and BAL cells and thirty thousand PBMC were acquired compensated. Compensation was set in BD FACS Diva Software (BD Biosciences). Unstained cells and single fluorochrome stained BD™CompBeads Compensation Particles (BD Biosciences) were used to set-up the machine. Flow cytometry was performed using a BD LSR II instrument (BD Biosciences). After incubation with Abs cells were washed and fixed with 2% paraformaldehyde. ![]() Cells were stained in a final volume of 100 µl FACS buffer (PBS, 2% FCS, 10 mM EDTA), for 30 min at 4 ☌. Nonspecific binding of antibodies (Abs) was eliminated by pre-incubating the cells in medium containing 10% normal human serum for 15 min at 4 ☌. Cells were incubated with “The LIVE⁄DEAD® Fixable Dead Cell Stain Kit” (Invitrogen) to exclude dead cells from the analysis. Adherent, in vitro polarized MDM were detached by incubation for 20 min with 1:1 mixture of 10 mM EDTA in PBS and RPMI 1640 media (PAA) at 37 ☌. PBMC and BAL cells from clinical samples and in vitro polarized MDM were used for flow cytometry analysis. 2.3 Experimental RV-induced asthma exacerbation study - subjects and clinical assessments Cell supernatants and RNA lysates were harvested at times indicated and stored at −80 ☌ (the 0 h time point means the time immediately after the 1 h incubation with RV, washing of non-adherent virus and adding fresh media). Cells were washed and any non-adherent virus was removed and re-suspended in the fresh media. Polarized or un-polarized MDM were then treated with live or UV-inactivated RV16 or RV1B MOI of 0.1, 0.5, 1, 2 for 1 h at room temperature. Un-polarized control MDM were maintained in culture overnight in MSFM alone. The mature MDM were then stimulated overnight with either 2 ng/mL of tumour necrosis factor (TNF) plus 20 ng/mL of IFN-γ (both from R&D Systems) or with 20 ng/mL of IL-4 (Invitrogen) to obtain MDM/TNF/IFNg and MDM/IL4 cells. The cells were differentiated for 7 days. ![]() An equal volume of fresh MSFM containing 10 ng/mL of human granulocyte-macrophage colony stimulating factor (GM-CSF, Invitrogen) and penicillin/streptomycin mixture (Invitrogen) were then added to the cells. Non-adherent cells were removed after 2 h of incubation at 37 ☌ in a humidified atmosphere containing 5% CO 2. The cells were washed, resuspended in Macrophage Serum Free media (MSFM, Invitrogen) and seeded into either 6-wells plates (NUNC) at 3 × 10 6 cells/well or Primaria™ Tissue Culture Dishes (Falcon®) at 30 × 10 6 cells/dish. ] by Ficoll-Hypaque density gradient centrifugation.
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