cd marker recognition of human cytokine-induced killer cells by flow cytometry

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

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 &#34;New Specimen&#34; 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 &#34;Load Sample&#34; 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.

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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
<ol>
	<li>On Day 0, culture the PBMCs (1 x 106) in fresh basal medium containing 1,000 IU/mL of IFN-&#947; for 24 h in a humidified cell culture incubator at 37 &#176;C and 5% CO2.</li>
	<li>On Day 1, refresh the medium with fresh basal medium containing 50 ng/mL of anti-CD3 antibody, 1 ng/mL of rh IL-1&#945;, and 1,000 U/mL of rh IL-2. Refresh the medium every 3 days.</li>
	<li>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).</li>
</ol>
2. Immunophenotyping for assessment of CIK cells
<ol>
	<li>Wash the CIK cells with 10 mL of sterile PBS. Centrifuge for 10 min at 300 x g and 18&#8722;20 &#176;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.</li>
	<li>Aliquot the CIK cells into six sterile 1.5 mL tubes at a density of ~5&#8211;10 x 105 cells/mL PBS. Label and treat as follows: Tube 1, Blank (no antibody); Tube 2, add 20 &#181;L of isotype IgG1-FITC; Tube 3, add 20 &#181;L of isotype IgG1-APC mAbs; Tube 4, add 20 &#181;L of CD3-FITC; Tube 5, add 20 &#181;L of CD56-APC mAbs; and Tube 6, add 20 &#181;L of CD3-FITC and 20 &#181;L of CD56-APC mAbs.</li>
	<li>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.</li>
	<li>Centrifuge the tubes for 10 min at 300 x g and 18&#8722;20 &#176;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.</li>
	<li>Repeat step 2.4.</li>
	<li>Leave the tubes in the dark before flow cytometric analysis.</li>
</ol>
3. CD marker recognition
<ol>
	<li>Transfer the cell suspension to a sterile 5 mL polystyrene round bottom tube with a cell strainer cap (100 &#956;m mesh) by gently pipetting through the cap. Put the tubes on the carousel in order.</li>
	<li>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.</li>
	<li>Create a scatter gating system for the CIK cell populations (Figure 1A).
	<ol>
		<li>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 &#62;5 x 104 to exclude cell debris.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Repeat step 3.3 for the investigation of all specimens.</li>
	<li>Open the files containing the statistical values of the individual specimen to analyze CIK cell populations and reprint them into analysis files.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>APC Mouse Anti-Human CD56 antibody</td>
			<td>BD</td>
			<td>555518</td>
			<td>B159</td>
		</tr>
		<tr>
			<td>APC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555751</td>
			<td>MOPC-21</td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Cytometer</td>
			<td>BD</td>
			<td>338962</td>
			<td>SN: R33896202856</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/F12</td>
			<td>HyClone</td>
			<td>SH30023.02</td>
			<td>Basal medium for OC-3 cell culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>For K562 and OC-3 cell culture. Complete medium contains 10% of FBS</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare Life Sciences</td>
			<td>71101700-EK</td>
			<td>Density gradient solution</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD3 antibody</td>
			<td>BD</td>
			<td>555332</td>
			<td>UCHT1</td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555748</td>
			<td>MOPC-21</td>
		</tr>
		<tr>
			<td>Human anti-CD3 mAb</td>
			<td>TaKaRa</td>
			<td>T210</td>
			<td>OKT3 
			Add 2.5 mL of stock (1 mg/1 mL) to 50 mL of Induction medium. Storage stock at -80 &#176;C</td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td>Basal medium for K562 cell culture. Storage stock at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

Source: Hsiao, C. H., et al. <a href="https://app.jove.com/v/60420">Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment.</a> J. Vis. Exp. (2020)
In this video, we demonstrate a flow cytometry-based technique to determine the proportion of cytokine-induced killer T-cells, CIK cells, expanded from a peripheral blood mononuclear cell culture.

<img alt="Figure 1" src="/files/ftp_upload/21559/21559_Figure1.jpg" /> 
Figure 1: The proportion of CD3+CD56+&#160;T cells from a representative PBMC sample.&#160;(A) Lymphocytes were recognized by specific size and granularity. Selected single cell population for analysis by flow cytometry. (B) Statistical analysis of CIK expansion efficacy from three healthy donors was conducted using a t-test (*, p &#60; 0.01).

Source: Hsiao, C. H., et al. Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment. J. Vis. Exp. (2020)
In this video, ...

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.

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

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