CD Marker Recognition of Human Cytokine-Induced Killer Cells by Flow Cytometry

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.

Begin this procedure with the preparation of CIK cells, as described in the text protocol. Then, wash the CIK cells with 10 milliliters of sterile PBS. Centrifuge for 10 minutes at 300 times g and 18 to 20 degrees Celsius. Aspirate the supernatant, and resuspend the cells with 5 milliliters of PBS. Count the cell numbers and test cell viability using the trypan blue exclusion assay.

Aliquot the CIK cells into six sterile 1.5-milliliter tubes at a density of approximately 0.5 to 1 million cells per milliliter of PBS. Label and treat the cells, as detailed in the text protocol. Gently mix the CIK cells with the antibodies by gently pipetting up and down at least three times with a 1-milliliter sterile pipette. Incubate the cells for 15 minutes at room temperature in the dark.

Centrifuge the tubes for 10 minutes at 300 times g and 18 to 20 degrees Celsius. Following centrifugation, aspirate the supernatant, and resuspend the cell pellet with 1 milliliter of PBS. Then, gently pipette the cells up and down at least three times with a 1-milliliter sterile pipette. Transfer the cell suspension to a sterile 5-milliliter polystyrene round-bottom tube with a cell strainer cap by gently pipetting through the cap. Then, place the tubes on the carousel in order.

Open the flow cytometry analysis software and create an experimental folder. Then, click the "New Specimen" button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol.

To create a scatter-gating system for the CIK cell populations, first, select tube 1 and click on the Dot Plot button to create an FSC-A/SSC-A plot. Draw a rectangle gate over the entire cell population with an FSC-A threshold greater than 5,000 to exclude cell debris.

Select the FSC-A/FSC-H parameter for the new dot plot, and draw a polygon gate around all single cells. Select the count versus FITC-conjugated (CD3) and count versus APC-conjugated (CD56) parameter for the new histogram plot. Select the FITC-conjugated (CD3) versus APC-conjugated (CD56) parameter for the new dot plot, and draw a four-quadrant gate to define the four subpopulations.

Record the data from 20,000 single cells in each specimen. Click the "Load Sample" button to analyze the blank control sample first. Identify the whole CIK cell population by using the CD56 and CD3 channel parameters. Open the files containing the statistical values of the individual specimen to analyze CIK cell populations and reprint them into analysis files.