Name: an in vitro assay to study the cytotoxic capability of cytokine-induced killer cells
Uploaded: 2023-11-30T12:47:42.000+00:00
Duration: 1 min 27 s
Description: All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

an in vitro assay to study the cytotoxic capability of cytokine-induced killer cells

An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells

To perform the cytotoxic assay, co-culture CIK and human chronic myeloid leukemia K562 cells, by adding 1 milliliter of K562 cells to each well in a 6-well plate at a density of 0.5 million cells per milliliter. Then, add 1 milliliter of basal medium with or without CIK cells to the 6-well plate, as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times.
Place the plate in the incubator for 24 hours. Now, co-culture CIK and ovarian OC-3 cells by adding 1 milliliter of basal medium with or without CIK cells to the 6-well plate, as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times with the blade in the incubator for 24 hours. Following incubation, harvest the CIK-K562 cell suspension directly into a 15-milliliter sterile tube.
To harvest both the suspension and adherent cells from the CIK-OC-3 groups, first, transfer the cell suspension to a 15-milliliter sterile tube. Wash the well with 1 milliliter of sterile PBS. Collect the PBS, and add it to the tube. Then, add 0.5 milliliters of cell dissociation enzyme solution, and incubate for five minutes at 37 degrees Celsius.
Add 1 milliliter of the solution from the same tube to the corresponding well, and gently mix the cells by pipetting up and down at least three times with a 1-milliliter sterile pipette. Collect all the cells in the same tube. Centrifuge at 300 x g for 10 minutes. Aspirate the supernatant, and resuspend the cells in 1 milliliter of sterile PBS. Pellet the cells at 300 x g for 10 minutes. Then, aspirate the supernatant, and resuspend the cells in 100 microliters of sterile PBS.
Add 5 microliters of 7-AAD dye to the cell suspension. Gently mix the cells by pipetting up and down at least three times with a 1-milliliter sterile pipette. Incubate for 10 minutes, and leave in the dark before analysis.
To perform the cytolytic capability assay, mix the cell suspension and repeat the preparation for flow cytometry. 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 cytolytic assay, first, select Tube 1 and click on the Dot Plot button. To create an FSC-A/SSC-A plot, draw a rectangle gate over all events with an FSC-A threshold greater than 50,000 to exclude cell debris. Select the SSC-A/CFSE parameter for the new dot plot. Then, select the 7-AAD/CFSE parameter for the new dot plot, and draw a four-quadrant gate to define the four subpopulations.
Click the Load Sample button to analyze the blank control sample first. Adjust the voltage of SSC-A and FSC-A. Identify the dead cell population by using the CFSE and 7-AAD channel parameters. Record the data from greater than 20,000 CFSE-positive cells in each specimen. Open the files containing the statistical values of each individual specimen to analyze the non-viable cell populations, and export the data 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. Preparation of materials
<ol>
	<li>Store reagents, antibodies, and chemicals as shown in the Material Safety Data Sheet (MSDS). Dissolve the drugs or cytokines in solvents as stock solutions and then aliquot for storage at -20 &#176;C or -80 &#176;C. 
	NOTE: Detailed information for material preparation is noted in the Table of Materials.</li>
</ol>
2. Peripheral blood mononuclear cell (PBMC) isolation
<ol>
	<li>Warm the density gradient solution (Table of Materials) to 18&#8211;20 &#176;C before use. Invert the solution bottle several times to ensure thorough mixing.</li>
	<li>Collect 3&#8722;5 mL of human venous blood sample in a heparinized vial and mix well by gently inverting the tube several times.</li>
	<li>Prepare 4 mL of density gradient solution in a 15 mL sterile tube.</li>
	<li>Carefully layer 1 mL of the blood sample onto the density gradient solution.</li>
	<li>Centrifuge at 400 x g for 30 min at 18&#8722;20 &#176;C (turn off the break).</li>
	<li>Carefully and immediately aspirate the buffy coat layer of mononuclear cells (about 1 mL) at once to avoid disturbing the layers to a sterile 15 mL tube using a 1 mL sterile pipette.</li>
	<li>Add at least 3 volumes (~3 mL) of phosphate-buffered saline (PBS) to the buffy coat in the centrifuge tube. Suspend the cells by gently pipetting them up and down at least 3x with a sterile pipette.</li>
	<li>Centrifuge at 400 x g for 10 min at 18&#8722;20 &#176;C. Aspirate the supernatant.</li>
	<li>Suspend the cell pellet with 5 mL of basal medium (Table of Materials) and transfer into a flask. Culture the cells in a cell culture incubator at 37 &#176;C and 5% CO2.</li>
</ol>
3. 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 recombinant human (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>
4. 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 pipet them up and down at least 3x with a 1 mL sterile pipette.</li>
	<li>Repeat step 4.4.</li>
	<li>Leave the tubes in the dark before flow cytometric analysis.</li>
</ol>
5. 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) parameter 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 5.3 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>
6. Culturing and staining of human chronic myeloid leukemia K562 cells and ovarian cancer OC-3 cells
<ol>
	<li>K562 cells
	<ol>
		<li>Culture K562 cells in complete media (RPMI basal medium containing 10% fetal bovine serum [FBS] and 50 U/mL antibiotics and adjust glucose to 4.5 g/L) at a density of 0.5&#8722;1 x 106 cells/mL in a cell culture flask and incubate in a humidified incubator at 37 &#176;C and 5% CO2.</li>
		<li>Transfer the culture media containing the K562 cells into 50 mL sterile tubes and pellet the cells at 300 x g for 10 min at 18&#8722;20 &#176;C on the day of the experiment.</li>
		<li>Aspirate the supernatant, resuspend the cells in 5 mL of sterile PBS, and mix well gently.</li>
		<li>Pellet the cells at 300 x g for 10 min. Aspirate the supernatant, resuspend the cells in PBS, and adjust the K562 cells to a concentration of 0.5-1 x 106 cells/mL.</li>
		<li>Add 0.5 &#181;L of carboxyfluorescein succinimidyl ester (CFSE) dye to the 1 mL of K562 cell suspension in a 15 mL sterile tube at a final concentration of 5 &#956;M. Gently mix the suspension by pipetting up and down at least 3x.</li>
		<li>Leave the tube in a cell culture incubator at 37 &#176;C and 5% CO2 for 10&#8211;15 min.</li>
		<li>Add 9 mL of PBS to the tube and pellet the cells at 300 x g for 10 min. Decant the supernatant and then suspend the cell pellet in 10 mL of complete media. Transfer the cell suspension to a cell culture flask and place in the incubator.</li>
	</ol>
	</li>
	<li>OC-3 cells
	<ol>
		<li>Culture OC-3 cells in complete media (DMEM/F12 medium containing 10% FBS and 50 U/mL antibiotics) at a density of 0.5&#8211;1 x 106 cells in a cell culture flask at 37 &#176;C and 5% CO2.</li>
		<li>Aspirate the culture media and wash the cells with PBS 1 day before the experiment.</li>
		<li>Detach the cells by adding 1 mL of cell dissociation enzyme solution (Table of Materials) and incubate for 5 min at 37 &#176;C.</li>
		<li>Suspend the cells by adding 5 mL of PBS and mix well gently. Pellet the cells at 300 x g for 10 min and aspirate the supernatant. Resuspend the cells in PBS and adjust the cells to a concentration of 0.5&#8211;1 x 106 cells/mL.</li>
		<li>Add 0.5 &#181;L of CFSE dye to 1 mL of the OC-3 cell suspension in a 15 mL sterile tube at a final concentration of 5 &#956;M. Gently mix the suspension by pipetting up and down at least 3x.</li>
		<li>Leave the tube in a cell culture incubator at 37 &#176;C and 5% CO2 for 10&#8211;15 min.</li>
		<li>Add 9 mL of PBS to the tube and pellet the cells at 300 x g for 10 min. Decant the supernatant and then suspend the cell pellet with complete media. Seed 5 x 105 cells/well into a 6 well plate and incubate in a humidified incubator at 37 &#176;C and 5% CO2 overnight.</li>
	</ol>
	</li>
</ol>
7. Cytotoxic assay
<ol>
	<li>Coculture of CIK and K562 cells (CIK-K562)
	<ol>
		<li>Count the K562 cells from step 6.1.7 and test the cell viability by trypan blue exclusion assay. Add 1 mL of K562 cells to each well in a 6 well plate at a density of 5 x 105/mL.</li>
		<li>Add 1 mL of basal medium with or without CIK cells from step 3.4 to the 6 well plate from step 7.1.1 as follows: Well 1 = Blank, K562 cells alone (5 x 105); Well 2 = CFSE-stained K562 cells alone (5 x 105); Well 3 = CIK cells (E [effector], 25 x 105) + CFSE-stained K562 cells (T [target], 5 x 105); Well 4 = CIK cells (E, 50 x 105) + CFSE-stained K562 cells (T, 5 x 105).</li>
		<li>Mix the cell suspensions by gently pipetting them up and down at least 3x. Place the plate in the incubator for 24 h.</li>
	</ol>
	</li>
	<li>Coculture of CIK and OC-3 cells (CIK-OC-3)
	<ol>
		<li>Add 1 mL of basal medium with or without CIK cells from step 3.4 to the 6 well plate from step 6.2.7 as follows: Well 1 = Blank, OC-3 cells alone (5 x 105); Well 2 = CFSE-stained OC-3 cells alone (5 x 105); Well 3 = CIK cells (E, 25 x 105) + CFSE-stained OC-3 cells (T, 5 x 105); Well 4 = CIK cells (E, 50 x 105) + CFSE-stained OC-3 cells (T, 5 x 105).</li>
		<li>Mix the cell suspensions by gently pipetting them up and down at least 3x. Put the plate in the incubator for 24 h.</li>
	</ol>
	</li>
	<li>7-Aminoactinomycin D (7-AAD) dye staining
	<ol>
		<li>Harvest the CIK-K562 cell suspension from step 7.1.3 directly into a 15 mL sterile tube.</li>
		<li>Harvest both the suspension and adherent cells from the CIK-OC-3 groups from step 7.2.2.
		<ol>
			<li>Transfer the cell suspension to a 15 mL sterile tube. Wash the well with 1 mL of sterile PBS, collect the PBS, and add to the tube. Add 0.5 mL of cell dissociation enzyme solution, and incubate for 5 min at 37 &#176;C.</li>
			<li>Add 1 mL of the solution from the same tube to the corresponding well and gently mix the cells by pipetting them up and down at least 3x with a 1 mL sterile pipette. Collect all the cells in the same tube.</li>
		</ol>
		</li>
		<li>Centrifuge at 300 x g for 10 min, aspirate the supernatant, and resuspend the cells in 1 mL of sterile PBS. Pellet the cells at 300 x g for 10 min, aspirate the supernatant, and resuspend cells in 100 &#181;L of sterile PBS.</li>
		<li>Add 5 &#181;L of 7-AAD dye (50 ng/&#181;L stock) to the cell suspension. Gently mix the cells by pipetting them up and down at least 3x with a 1 mL sterile pipette. Incubate for 10 min and leave in the dark before analysis.</li>
	</ol>
	</li>
	<li>Cytolytic capability assay
	<ol>
		<li>Mix the cell suspension from step 7.3.4 and repeat steps 5.1 and 5.2 once.</li>
		<li>Click the New Specimen button to add a specimen and tube to the experiment and name the tubes as follows: Tube 1, K562 (or OC-3) cells only; Tube 2, CFSE-stained K562 (or OC-3) cells only; Tube 3, E:T = 5:1; Tube 4, E:T = 10:1.</li>
		<li>Create a Scatter Gating System for the cytolytic assay (Figure 2A).
		<ol>
			<li>Select Tube 1 and click on the Dot Plot button to create an FSC-A/SSC-A plot. Draw a rectangle gate over all events with an FSC-A threshold &#62;5 x 104 to exclude cell debris.</li>
			<li>Select the SSC-A/CFSE parameter for the new dot plot. Select the 7-AAD/CFSE parameter for the new dot plot and draw a four-quadrant gate to define the four subpopulations.</li>
			<li>Click the Load Sample button to analyze the blank control sample first.</li>
			<li>Adjust the voltage of SSC-A and FSC-A. Identify the dead cell population by using the CFSE and 7-AAD channel parameters. Record the data from &#62;20,000 CFSE+ cells in each specimen.</li>
		</ol>
		</li>
		<li>Repeat section 7.4.6 for the investigation of all specimens.</li>
		<li>Open the files containing the statistical values of each individual specimen to analyze the non-viable cell populations and export the data into analysis files.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7-Amino Actinomycin D</td>
			<td>BD</td>
			<td>559925</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD56 antibody</td>
			<td>BD</td>
			<td>555518</td>
			<td>B159</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555751</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Cytometer</td>
			<td>BD</td>
			<td>338962</td>
			<td></td>
			<td>SN: R33896202856</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>BD</td>
			<td>565082</td>
			<td></td>
			<td>Reconstritution of CFSE dye (500 mg) with 90 mL of DMSO</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>SIGMA</td>
			<td>G8644</td>
			<td></td>
			<td>For K562 cell culture. Add 12.5 mL to 500 mL of complete medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/F12</td>
			<td>HyClone</td>
			<td>SH30023.02</td>
			<td></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></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></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>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555748</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-CD3 mAb</td>
			<td>TaKaRa</td>
			<td>T210</td>
			<td>OKT3</td>
			<td>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>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
			<td>Add 5 mL of stock (10,000 U/mL) to 500 mL of complete medium. Storage stock at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Proleukin</td>
			<td>NOVARTIS</td>
			<td></td>
			<td></td>
			<td>Reconstitution of Proleukin Powder (22x106 IU) with 1.2 mL of sterile water and add 2.7 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interferon-gamma</td>
			<td>CellGenix</td>
			<td>1425-050</td>
			<td></td>
			<td>Reconstitution of rh IFN-g (5x105 IU/50 &#181;g) with 200 &#181;L of sterile water and add 20 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-1 alpha</td>
			<td>PEPROTECH</td>
			<td>200-01A</td>
			<td></td>
			<td>Reconstitution rh IL-1&#945; (10 &#181;g) with 1 mL of sterile water and add 5 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td></td>
			<td>Basal medium for K562 cell culture. Storage stock at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sigma 3-18K Centrifuge</td>
			<td>Sigma</td>
			<td>10295</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td></td>
			<td>Cell dissociation enzyme; For deattachment of adheren cells. Storage at room temperature</td>
		</tr>
		<tr>
			<td>X-VIVO 15 medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
			<td>Basal medium for PBMC and CIK cells. Storage 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).
This video demonstrates an in vitro technique to measure the cytotoxic ability of cytokine-induced killer (CIK) cells against cancer cells. Upon co-culturing target cancer cells with CIK cells, the dead cancer cells are identified using a fluorescent viability dye and are quantified using flow cytometry, estimating the cytotoxic effect of CIK cells.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/21802/21802_Figure1.jpg" /> 
Figure 1: The proportion of CD3+CD56+ T cells from a representative PBMC sample. (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).
<p class="jove_conten...

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Take a culture of cancer cells with fluorescently labeled cytoplasmic proteins.
Add cytokine-induced killer or CIK cells &#8212; effector T cells with natural killer cell-like characteristics &#8212; possessing anti-tumor activity.
Natural killer group 2 member D or NKG2D&#160; receptors on the CIK cells bind to stress-inducible proteins on the cancer cells.
The binding induces CIK cells to release cytoplasmic granule toxins &#8212; perforins and granzymes.
Perforins form pores in the cancer cell membrane, allowing the entry of granzymes, which activate caspases, inducing apoptosis.
Post-incubation, harvest the cells. Add a fluorescent nucleic acid-binding cell viability dye.
The dye enters through the damaged cell membrane and binds to DNA, resulting in dual-stained dead cells.
With an intact cell membrane, live cells are impermeable to the dye.
Pass the cells through a flow cytometer to detect live cells displaying single fluorescence and dead cells with dual fluorescence.
Create a scatter plot to measure the percentage of dead cancer cells, quantifying the cytotoxic effect of CIK cells.

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This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

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Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

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Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells

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An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells