An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Begin with a recipient mouse pre-injected with genetically engineered, cell proliferative dye-stained CD8⁺ donor T lymphocytes. These lymphocytes express thymus-1.1 antigen marker, enabling their differentiation from the recipient T lymphocytes.

Subcutaneously inject endotoxin-free ovalbumin with a test adjuvant.

Adjuvants enhance the presentation of ovalbumin to antigen-presenting cells, or APCs, which process the ovalbumin and present the fragment with MHC.

Activated APCs migrate to the lymph nodes and spleen, interacting with pre-injected CD8⁺  T lymphocytes, differentiating and proliferating them into cytotoxic T lymphocytes.

Collect the mouse's lymph node and spleen, and generate a single-cell suspension.

Add a lysis buffer — exclusively lysing red blood cells.

Collect the lymphocytes and introduce a mix of fluorophore-labeled antibodies that interact with thymus-1.1, CD4, and CD8 marker-expressing cells.

Using flow cytometry, identify lymphocytes expressing thymus-1.1 and CD8, distinguishing them from recipient CD8⁺ T lymphocytes.

Quantify the green fluorescence of the proliferative dye from these cells. A reduction in fluorescence intensity confirms adjuvant-mediated cytotoxic T lymphocyte generation.