Whole blood was stimulated as previously described,26 (link) with little modification in relation to the used agonists. For each stimulation, 100 μL heparinized blood was treated with Polymyxin B, 10 µg/mL for 20 minutes (except the sample stimulated with lipopolysaccharide) and hereafter, incubated at 37°C in a 5% CO2 atmosphere for 1 hour with the individual agonists Pam3csk48 (Invivogen, San Francisco, CA; TLR1/2), Pam2csk4 (Invivogen; TLR2/6), lipopolysaccharide (Invivogen; TLR4), flagellin-ST (Invivogen; TLR5), CL097 (Invivogen; TLR7/8), ssRNA40 (LyoVec, Invivogen; TLR8) and with polymyxin A (Sigma-Aldrich, St. Louis, MO) or tumor necrosis factors a (TNFa; R&DSystems, Minneapolis, MN) as positive controls. After activation, the erythrocytes were lysed in EasyLyse (Dako) and washed twice in phosphate-buffered saline. The cells were incubated with anti-human CD62L or isotype control (BD Bioscience Pharmingen 555544 resp. 555749) for 20 minutes in 4°C.26 (link)
Flagellin st
Manufactured by InvivoGen
Flagellin-ST is a purified flagellin protein derived from Salmonella typhimurium. Flagellin is the principal structural component of bacterial flagella and is recognized by the innate immune system as a pathogen-associated molecular pattern (PAMP). Flagellin-ST functions as a Toll-like receptor 5 (TLR5) agonist, which can be used to stimulate the innate immune response in various applications.
Automatically generated - may contain errors
2 protocols using flagellin st
1
Whole Blood Stimulation and CD62L Expression
2
Inflammasome Activation in BMDMs
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For inflammasome activation, 5 × 105 BMDMs were plated in 24-well plates and primed with 100 ng ml−1 LPS-EK (Invivogen, San Diego, CA) for 4 h. Cells were then either treated with tumour homogenate in DMEM (Fig. 2h ) for 24 h or transfected with 500 ng Flagellin-ST (Invivogen) for 6 h. For cells treated with metformin, the indicated dose of metformin was added 1 h before transfection with flagellin. The media was collected at the end of the experiment and IL-1β was measured by ELISA. Antibody pairs for the ELISA were from (R&D Systems).
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