Tumor Necrosis Factor alpha (TNFa) (PE)

Human tumor necrosis factor alpha (TNF alpha) is a proinflammatory cytokine expressed mainly by activated monocytes and macrophages. TNF alpha exerts a key role with regard to pathogenesis of many infectious and inflammatory diseases. Applications: Suitable for use in Flow Cytometry, Western Blot, and Immunohistochemistry. Other applications not tested. Recommended Dilution Flow Cytomtery: 1:50 (5ug/ml) labels 5x10e5 cells Optimal dilutions to be determined by the researcher. Flow Cytometry Protocol: 1. Ficoll prepared human peripheral blood lymphocytes were stimulated by incubating 6 hours in the presence of 50ng/ml PMA, 1mM ionomycin and 1ug/ml brefeldin A. 2. Cells were then harvested, washed and fixed for 30 min with 2% buffered formaldehyde after which they were washed two times. 3. Fixed cells were permeabolized 10 minutes in a buffer containing 0.3% Saponin. (Subsequent reagent incubations and washes were done using this buffer.) 4. Five x 10e5 cells per tube were preincubated ~5 minutes with 20ul of 0.25mg/ml human IgG (To block non specific binding) after which they were incubated 45 minutes on ice with 80ul of T9160-29D at a dilution factor of 1:50 (5ug/ml). 5. Cells were washed three times, fixed and analyzed by FACS. 6. A net 27% sub population of the cells stained positive with a mean shift of 0.85 log10 fluorescent units when compared to a Mouse IgG1/R-PE negative control at a similar concentration. 7. Binding was blocked when pre incubated 10 minutes with 20ul of 0.5mg/ml anti-TNFa antibody (T9160-29A) Storage and Stability: May be stored at 4 degrees C before opening. DO NOT FREEZE! Stable at 4 degrees C as an undiluted liquid. Dilute only prior to immediate use. Stable for 12 months at 4 degrees C. Freezing R-Phycoerythrin (PE) conjugates will result in a substantial loss of activity. PE conjugates are sensitive to light.