By by Dieter Korholz (Editor), Wieland Kiess (Editor)
Hands-on laboratory specialists describe for the beginner investigator a bunch of novel applied sciences and molecular recommendations in particular designed to review mobile immunology and advertise its gene treatment purposes. offered in step by step aspect to make sure prepared reproducibility, those protocols diversity from movement cytometric options to observe cytokines and development elements in numerous specimens, to tools for producing and increasing dendritic cells and hematopoietic progenitors from assorted origins. even if the protocols require little prior event, even hugely expert researchers will increase their talents with many novel timesaving concepts.
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Extra resources for Cytokines and Colony Stimulating Factors: Methods and Protocols (Methods in Molecular Biology Vol 215)
3. Bicoll (Biochrome, Berlin, Germany), sterile, should be stored at 4–25°C and protected from light (storage in cold will increase the shelf life). 4. RPMI 1640 medium supplemented with the following: 10% heat-inactivated fetal calf serum (FCS) 10 U/mL penicillin 10 µg/mL streptomycin 2 mM L-glutamine This can be stored at 4°C up to 3 wk. 5. 0371 g EDTA; add 1 L with distilled water, sterilize, and store at room temperature. 2. Stimulation 1. 2. 3. 4. 96 Flat-bottom 96-well cell culture plates.
1. 2. 1. Add 120 µL lin 1 FITC, 60 µL anti-HLA-DR PerCP, and 30 µL CD11c APC to an empty 5-mL polypropylene round-bottom tube. 2. Add 300 µL whole blood (WB) to the antibody mixture and vortex. 3. Incubate for 15 min at RT in the dark. 4. Add 3 mL FACS Lysing Solution 1X working concentration, cap tubes, and vortex. 5. Incubate for 10 min at RT in the dark. 6. Centrifuge 7 min at 500g. 7. Aspirate supernatant carefully and vortex gently to break off the cell pellet. 8. Resuspend surface-stained cells in 1 mL FACS Permeabilizing Solution 1X working concentration.
Incubate for 2 h. The cell activation is performed at 37°C humidiﬁed atmosphere and 5% CO2. During incubation, cap tubes loosely to prevent evaporation of the sample, but to allow gas exchange. The use of polypropylene labware for DC activation is important because it increases the DC recovery. DCs can stick to polystyrene surfaces and may be lost for further analysis. 2. Blood Activation: Cytokine Kinetic Assay (TNF-α, IL-1-β, IL-6) For the determination of cytokine kinetics, LPS-activated whole blood is processed every hour in a time window from 0 to 8 h of incubation.