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    Protocols

Zellnet Consulting, Inc.  
(201) 346-0710  

info@zellnet.com  




Please, find below some commonly requested protocol recommendations.






General Elispot Assay Protocol

Day 1
Coat plate:
  1. Prewet PVDF plate (MSIPS4510; Millipore) with 15 ul 70% Ethanol, decant Ethanol and wash plate 3 x with 150 ul PBS.
  2. Coat IP plate with 100 ul capture antibody in PBS (1ug per well works well for most antibodies).
  3. Incubate overnight at 4oC.
Day 2
Prepare plate:
  1. Decant antibody solution.
  2. Wash plate once with 100 ul PBS.
  3. Block with 150 ul of T-cell medium (10% serum) for at least 2 hours at 37oC.

    Prepare effector cells:

  4. Purify immune cells of interest.
  5. Wash effector cells in T-cell medium, count and resuspend at a final concentration of 2-4x10^6 cells/ml in T-cell medium.

    Prepare target cells:

  6. Wash target cells 2x with T-cell medium.
  7. Pulse with peptide at appropriate concentration if necessary, and irradiate.
  8. Resuspend in T-cell medium at appropriate concentration.

    Plate out assay:

  9. Decant blocking medium.
  10. Gently plate out purified immune cells (recommendation: 1-2x10^5 cells/well) in 50 ul T-cell medium/well. Allow cells to settle down on membrane for 1-2 hours in incubator.
  11. Carefully add target cells in 50 ul T- cell medium/well.
  12. Incubate for 20 hours (IFN-gamma assay) in 37oC, 5% CO2.
Day 3
  1. Discard cells.
  2. Wash plate rigorously 6 x with PBS/0.05% Tween 20.
  3. Add 100 ul/well biotinylated detection antibody in PBS/0.5% BSA after filtering the antibody diluted in buffer with low-protein-binding filter, 0.22 um.
  4. Incubate for at least 2 hours at 37oC.
  5. Wash plate 4 x with PBS/0.05% Tween 20.
  6. Prepare Avidin-Enzyme-Complex (Vector Laboratories) about 30 minutes prior to use, and leave at room temperature.
  7. Add 100 ul of Avidin-Enzyme-Complex/ well.
  8. Incubate for 1 hour at room temperature.
  9. Discard Avidin-Enzyme-Complex, wash 2x with PBS/0.05% Tween 20, followed by 3x washes with plain PBS.
  10. Add 100 ul AEC substrate per well and incubate for 4 minutes.
  11. Stop spot development under tap water.
  12. Remove underdrain and all excess liquid from wells.
  13. Dry back of wells thoroughly with a paper towel.
  14. Let plate dry overnight in darkness before membrane removal with ELI-Puncher Kit (ZellNet Consulting).
  15. Count spots (recommendation: KS Elispot Automated Reader System from Carl Zeiss).





AEC preparation

  1. Add 1 tablet of AEC (Sigma) to 2.5 ml dimethylformamid (in 50 ml tube). Wait until completely dissolved (app. 5 minutes).
  2. In the meantime, prepare acetate buffer as follows:
    • 46.9 ml dd H2O
    • 4.6 ml 0.1 N acetic acid
    • 11 ml 0.1 M sodium acetate
  3. Add 47.5 ml of acetate buffer to dissolved AEC tablet.
  4. Add 25 ul 30% H2O2 and mix well.
  5. Filter through 0.45 um filter.
  6. Add 100 ul AEC solution per well.
















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