Encyclopedia of Experiments: Immunology
JoVE 비디오를 활용하시려면 도서관을 통한 기관 구독이 필요합니다. 전체 비디오를 보시려면 로그인하거나 무료 트라이얼을 시작하세요.
Transcript
Cytokine-induced killer, CIK, cells are heterogeneous immune cell populations generated ex vivo from peripheral blood mononuclear cell (PBMC) cultures stimulated with specific cytokines and monoclonal antibodies. CIK cells comprise cell populations, including CD3+CD56+ NK-T cells, CD3+CD56- T cells, and CD3-CD56+ NK cells.
To determine the CIK cell proportion, begin with tubes containing a PBMC-derived CIK cell suspension. Add anti-CD3 and anti-CD56 antibodies conjugated to different fluorophores and their mixture to designated tubes. Add isotype control antibodies conjugated to similar fluorophores as specific antibodies into other tubes.
Fluorescently-labeled anti-CD3 and anti-CD56 antibodies recognize and bind to CD3 and CD56 expressed on CIK cell populations, respectively. Isotype control antibodies lack target antigen specificity, facilitating non-specific background signal measurements.
Pipette cells into round-bottom tubes with cell strainer caps, that help in removing cell aggregates. Load the tubes into a flow cytometer carousel. During the run, each cell passes through the channel and encounters the laser beams, scattering the light.
One detector measures the forward scattered light intensity, indicative of cell size; another measures the side scattered light intensity perpendicular to the laser beam, suggestive of cell granularity. Together, this information helps identify the immune cell population.
Simultaneously, laser beams of specific excitation wavelengths excite the fluorophore-conjugated antibodies on the cells, causing fluorescent signal emissions which are detected.
The analysis of specific fluorescent intensities on the cells determines the proportion of CIK cell subpopulations.
