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

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. CIK induction and expansion

1. On Day 0, culture the PBMCs (1 x 10⁶) in fresh basal medium containing 1,000 IU/mL of IFN-γ for 24 h in a humidified cell culture incubator at 37 °C and 5% CO₂.
2. On Day 1, refresh the medium with fresh basal medium containing 50 ng/mL of anti-CD3 antibody, 1 ng/mL of rh IL-1α, and 1,000 U/mL of rh IL-2. Refresh the medium every 3 days.
3. On Day 7, refresh the medium with fresh basal medium containing 1,000 U/mL of rh IL-2. Refresh the medium every 3 days until the end of cell expansion (Day 14).

2. Immunophenotyping for assessment of CIK cells

1. Wash the CIK cells with 10 mL of sterile PBS. Centrifuge for 10 min at 300 x g and 18−20 °C, aspirate the supernatant and resuspend the cells with 10 mL of PBS. Count the cell number and test cell viability using the trypan blue exclusion assay.
2. Aliquot the CIK cells into six sterile 1.5 mL tubes at a density of ~5–10 x 10⁵ cells/mL PBS. Label and treat as follows: Tube 1, Blank (no antibody); Tube 2, add 20 µL of isotype IgG1-FITC; Tube 3, add 20 µL of isotype IgG1-APC mAbs; Tube 4, add 20 µL of CD3-FITC; Tube 5, add 20 µL of CD56-APC mAbs; and Tube 6, add 20 µL of CD3-FITC and 20 µL of CD56-APC mAbs.
3. Gently mix the CIK cells with the antibodies by gently pipetting them up and down at least 3x with a 1 mL sterile pipette, and then incubate for 30 min at room temperature in the dark.
4. Centrifuge the tubes for 10 min at 300 x g and 18−20 °C. Aspirate the supernatant and suspend the cell pellet once with 1 mL of PBS. Gently pipette them up and down at least 3x with a 1 mL sterile pipette.
5. Repeat step 2.4.
6. Leave the tubes in the dark before flow cytometric analysis.

3. CD marker recognition

1. Transfer the cell suspension to a sterile 5 mL polystyrene round bottom tube with a cell strainer cap (100 μm mesh) by gently pipetting through the cap. Put the tubes on the carousel in order.
2. Open the flow cytometry analysis software and create an experimental folder. Click the New Specimen button to add a specimen and tube to the experiment and name the tubes as follows: Tube 1, Blank; Tube 2, Isotype IgG1-CD3; Tube 3, Isotype IgG1-CD56; Tube 4, CD3; Tube 5, CD56; Tube 6, CD3CD56.
3. Create a scatter gating system for the CIK cell populations (Figure 1A).
   1. Select Tube 1 (Blank) 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 >5 x 10⁴ to exclude cell debris.
   2. Select the SSC-A/SSC-H parameter for the new dot plot and draw a polygon gate around all single cells. Select the Count/FITC (CD3) and Count/APC (CD56) parameters for the new histogram plot, respectively. Select the FITC (CD3)/APC (CD56) parameter for the new dot plot and draw a four-quadrant gate to define the four subpopulations.
   3. 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.
4. Repeat step 3.3 for the investigation of all specimens.
5. Open the files containing the statistical values of the individual specimen to analyze CIK cell populations and reprint them into analysis files.