By Dikran N Dikranjan

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5 Optimal plate setup for testing of three different PBMC samples against three different peptide pool stimulants, one control peptide pool, and a mitogenic control. A reference sample is also included, which only needs to be tested against the control peptide pool and mitogen for efficient trending information clean outside of vial with ethanol wipe, and pipet the content into a 50 mL sterile conical tube. 5. Dropwise and slowly add warm assay test medium (see Note 9) supplemented with 50 U/mL benzonase to make the sample up to 10 mL (see Note 15).

Use an automated ELISpot imaging system for spot counting (different automated reader systems are available with different capabilities and software features) (see Note 26). 2. Adjust reading parameters by comparing negative control wells and wells with spots. Spots from antigenic stimulation wells are preferred over positive control spots, which may have a different appearance due to different kinetics of cytokine induction and secretion. Depending on the reader and software used, settings can typically be adjusted to all or some of the parameters related to the following spot features: size, color, intensity of staining, shape, and fading of color from spot center to its periphery.

Kutscher S, Dembek CJ, Deckert S, Russo C, Korber N, Bogner JR, Geisler F, Umgelter A, Neuenhahn M, Albrecht J, Cosma A, Protzer U, Bauer T (2013) Overnight resting of PBMC changes functional signatures of antigen specific T-cell responses: impact for immune monitoring within clinical trials. PLoS One 8(10), e76215. 0076215 36. Mascotti K, McCullough J, Burger SR (2000) HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40(6):693–696. 1046/j. x 37. Lenders K, Ogunjimi B, Beutels P, Hens N, Van Damme P, Berneman ZN, Van Tendeloo VF, Smits EL (2010) The effect of apoptotic cells on virus-specific immune responses detected using IFN-gamma ELISPOT.

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