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Eicosanoid and cytokine analysis

AL Antonie Lechner
JE Julia Esser-von Bieren

Last updated date: Aug 2, 2021 Views: 752 Forks: 0

original An abbreviated version of this protocol was published in Science Translational Medicine in Apr, 2020
An anti-inflammatory eicosanoid switch mediates the suppression of type-2 inflammation by helminth larval products
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cysLT ELISA

  1. Prepare Buffers
    • Immunoassay Buffer A Preparation: Dilute contents of one vial of Immunoassay Buffer A Concentrate (10X) with 90ml Milli-Q water. Make sure to rinse the vial many times to remove any salts that have precipitated.
    • Wash Buffer Preparation: Dilute 2.5ml Wash Buffer Concentrate (400X) to a total volume of 1l with Milli-Q water and add 0.5ml of Polysorbate-20
    • Store all diluted buffers at 4°C
  2. Dilute standards and samples and incubate plate
    • Place samples on ice to thaw
    • Fill a 15ml falcon tube with Milli-Q water
    • cysLT AChE Tracer: reconstitute 100 dtn cysLT AChE Tracer with 6ml Immunoassay Buffer
    • cysLT ELISA Monoclonal Antibody: reconstitute 100 dtn cysLT ELISA AB with 6 ml Immunoassay Buffer A
    • Sample preparation and dilution 1:25
        • Label a round bottom 96-well plate
        • Add Milli-Q water, add sample according to plan and mix by carefully pipeting up and down
        • In order to make sample transfer quicker, later also pipet 200µl of each standard on plate
    • cysLT EIA Standard: Equilibrate a pipette tip in ethanol by repeatedly filling and expelling the tip with EtOH several times. Using the equilibrated pipette tip, transfer 100μl of the cysLT EIA Standard into a clean tube, then dilute with 900μl Milli-Q water
    • Preparation of standards: Label eight clean tubes #1 through #8. Aliquot 900μl Milli-Q water to tube #1 and 500μl Milli-Q H2O to tubes #2-8. Transfer 100μl of the bulk standard (25 ng/ml) to tube #1 and mix thoroughly. Serially dilute the standard by removing 400μl from tube #1 and placing in tube#2; mix thoroughly. Next, remove 400μl from tube #2 and place it into tube #3; mix thoroughly. Repeat for tubes #4-8. Stable for max 2h!!! (Make last thing before sample dilutions go on plate!)
    • 2 wells for blank, 2 wells for Non Specific Binding, 3 wells for B0 and 1 well for Total Activity (TA) have to be included
    • Add 50µl Milli-Q to NSB and B0 wells
    • Add 50µl Immunoassay Buffer A to NSB wells
    • Add standards to appropriate wells, starting with S8, using same and newly equilibrated pipette tip
    • Transfer 50µl of each sample to ELISA plate using multichannel
    • Add 50ml cysLT tracer to each well except TA and Blk
    • Add 50µl of cysLT ELISA AB to each well except TA, NSB and Blk immediately
    • Cover plate with plastic film and incubate overnight at 4°C (orbital shaker from biochemistry lab with a plate for balance)
  3. Develop and read plate
  • Development:
    • Reconstitut Ellman’s Reagent immediately before use with 20ml of Milli-Q; protect from light when not in use
    • Empty wells and rinse x5 with Wash Buffer
    • Add 200 μl of Ellman’s Reagent to eachwell
    • Dilute cysLT Tracer 1:10 with ELISA Buffer(5μl of Tracer into 450μl Immunoassay Buffer)and add 5μl to TA well
    • Cover plate with plastic film and incubate at RTC on orbital shaker in the dark for 90-120min Reading of plate
  • Wipe bottom of plate with a clean tissue and remove plate cover being careful to keep Ellman’s Reagent from splashing on the cover.
  • Read the plate at 405nm x5 at 90min and again x5 at 120min
  • Load 120min data (.txt format using dots not commas) into online platform MyAssays.com and analyze
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Lechner, A and Esser-von Bieren, J(2021). Eicosanoid and cytokine analysis. Bio-protocol Preprint. bio-protocol.org/prep1323.
  2. Jiménez, M. D. L. R., Lechner, A., Alessandrini, F., Bohnacker, S., Schindela, S., Trompette, A., Haimerl, P., Thomas, D., Henkel, F., Mourão, A., Geerlof, A., Costa, C. P. D., Chaker, A. M., Brüne, B., Nüsing, R., Jakobsson, P., Nockher, W. A., Feige, M. J., Haslbeck, M., Ohnmacht, C., Marsland, B. J., Voehringer, D., Harris, N. L., Schmidt-Weber, C. B. and Bieren, J. E.(2020). An anti-inflammatory eicosanoid switch mediates the suppression of type-2 inflammation by helminth larval products . Science Translational Medicine 12(540). DOI: 10.1126/scitranslmed.aay0605

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