IFNγ ELISPOT Assay for M. tuberculosis

IFNγ ELISPOT (MABtech, Cincinnati, OH) assays were performed as per manufacturer instructions and previously described^{39 (link)}. Briefly, PBMCs and neutrophils were obtained from macaques as previously indicated and three cell suspensions were added to duplicate wells: neutrophils only (125,000 cells/well), PMBCs (125,000 cells per well) and neutrophils with PBMCs (1:1 ratio, total 250,000 cells per well). Cells were stimulated with CFP or a cocktail of M. tuberculosis ESAT-6 and 38.1 peptides as previously described^{8 (link)} and incubated overnight at 37°C with 5% CO₂. T cell viability after this treatment was experimentally confirmed and not found to be impaired by overnight co-culture with CFP-stimulated neutrophils. Plates were read with a CTL SPOT reader (Immunospot, Cleveland, OH) and analyzed for the number of spots per well, spot size, and signal intensity per spot.

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Gideon H.P., Phuah J., Junecko B.A, & Mattila J.T. (2019). Neutrophils express pro- and anti-inflammatory cytokines in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques. Mucosal immunology, 12(6), 1370-1381.

Publication 2019

IFNγ ELISPOT

Assays Cell viability Cells Co culture Elispot Ifnγ M tuberculosis Macaques Neutrophils Peptides Spots

Corresponding Organization : University of Pittsburgh

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Variable analysis

independent variables

Stimulation condition (CFP or M. tuberculosis ESAT-6 and 38.1 peptides)

dependent variables

Number of spots per well
Spot size
Signal intensity per spot

control variables

Cell types (neutrophils only, PBMCs, neutrophils with PBMCs)
Cell counts (125,000 cells/well for neutrophils or PBMCs, 250,000 cells/well for neutrophils with PBMCs)
Incubation time (overnight)
Incubation temperature (37°C)
Incubation atmosphere (5% CO2)
T cell viability (confirmed to not be impaired by overnight co-culture with CFP-stimulated neutrophils)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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