Home › Immunology › Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle
Immunology JoVE (Open Access) Citable · DOI

Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

DOI: 10.3791/52833-v
What you'll learn
  • ✓ Perform long-term culture of bovine PBMCs with mycobacterial antigens and IL-2
  • ✓ Execute IFN-γ ELISPOT assay protocol across three-day timeline
  • ✓ Differentiate central memory T cell responses via flow cytometry analysis
  • ✓ Measure anti-mycobacterial vaccine protective immunity in cattle
Protocol

Biopharma Insights Long-term cultured interferon-γ enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Difficulty
advanced
Total time
~16 days (14-day culture + 3-day ELISPOT assay + analysis)
Model organism
Cattle (bovine peripheral blood mononuclear cells)
Biosafety
BSL-2

Steps

1
Establish long-term PBMC culture with antigens

Stimulate peripheral blood mononuclear cells with mycobacterial antigens and interleukin-2 for 14 days to enable central memory T cell expansion and differentiation.

▶ 01:40
2
Prepare ELISPOT plate coating and blocking

Coat ELISPOT plates with capture antibodies and apply blocking solution on day 1 of the assay protocol.

▶ 03:38
3
Add cultured cells to ELISPOT plates

Transfer stimulated PBMCs to prepared ELISPOT plates and incubate overnight to allow IFN-γ secretion and capture.

▶ 04:30
4
Isolate adherent cells from culture

Harvest and separate adherent cell populations from long-term cultured cells for downstream analysis.

▶ 05:15
5
Prepare samples for flow cytometry analysis

Aliquot cultured cells and stain with fluorescent antibodies for phenotypic characterization of T cell subsets.

▶ 05:37
6
Harvest cultured cells on day thirteen

Collect and prepare long-term cultured PBMC populations prior to final ELISPOT assay processing.

▶ 06:15
7
Add detection antibodies and enzyme substrate

Apply alkaline phosphatase-conjugated secondary antibodies and substrate solution to ELISPOT plates on day 3.

▶ 09:05
8
Prepare alkaline phosphatase enzyme solution

Dilute and prepare alkaline phosphatase-conjugated detection reagent for ELISPOT plate development.

▶ 10:05
9
Prepare and apply substrate solution

Mix substrate components and apply to ELISPOT plates to develop colorimetric signal at IFN-γ-secreting cell sites.

▶ 11:08
10
Scan and quantify ELISPOT plate spots

Use automated ELISPOT reader to image plates and count IFN-γ-secreting spot-forming cells.

▶ 11:55
11
Perform flow cytometry characterization

Analyze phenotypic markers on cultured T cells using multiparameter flow cytometry to identify memory subsets.

▶ 12:12
12
Interpret results and assess vaccine response

Correlate IFN-γ ELISPOT data with flow cytometry findings to measure central memory responses and predict anti-mycobacterial protective immunity.

▶ 12:25
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