determining the role of neutrophil-like cells in lymphoma cell sensitivity to a test drug

Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug

To set up a 3D co-culture, add 50,000 RL lymphoma B cells alone, or mix with HL60-differentiated cells in sterile 15-milliliter tubes.
Spin down the samples and resuspend the pellets in 300 microliters of basement membrane matrix using a 1-milliliter pipette tip with the opening cut to 2 to 3 millimeters. Next, incubate 300 microliters of the cells in each well of a 24-well plate for 30 minutes at 37 degrees Celsius and 5% CO2.
At the end of the incubation, add 1 milliliter of complete RPMI medium to each well, and return to 3D cultures to the incubator for another seven days with medium changes every two days. On day 5, add 10 nanomolar vincristine to the cultures.
After one week of culture, aspirate the medium and wash each well 2 times with 1 milliliter of ice-cold PBS. Then, add 3 milliliters of ice-cold PBS with EDTA to each well, and scrape the bottom of the wells with a 200-microliter pipette tip to detach the gels. Shake the plate gently on ice for 30 minutes. Then, transfer the cell suspensions to individual sterile 15-milliliter tubes for gentle shaking on ice for another 30 minutes.
Confirm the appearance of a homogeneous cell suspension. Then, spin down the cells, followed by a PBS wash, and resuspend the pellets in fresh PBS with FBS. Finally, label the cells with the appropriate antibodies and viability dyes as just demonstrated, and analyze the RL and HL60-differentiated cells by flow cytometry using the illustrated gating strategy.

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1. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model
<ol>
	<li>Differentiation of Human Promyelocytic (HL60) Cells into Neutrophil-like Cells
	<ol>
		<li>Adjust HL60 cell number to 3 x 105 cells/ml then add 1 &#181;M retinoic acid (RA) and 1.25% DMSO to induce their differentiation. Seed each 3 x 105&#160;HL60 cells per well in 48-well plate then incubate for 48 hr at 37 &#176;C with 5% CO2.</li>
		<li>Later, collect the cells and centrifuge at 300 g for 5 min at RT. Resuspend the cells with complete RPMI medium for counting using cell viability counter.</li>
		<li>Then dilute cell suspension in complete RPMI medium to adjust HL60 cell number to 3 x 105&#160;cells/ml and induce again their differentiation by adding 1 &#181;M retinoic acid (RA) and 1.25 % Dimethyl sulfoxide (DMSO). Seed each 3 x 105&#160;HL60 cells per well in 48-well plate and incubate for another 48 hr at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Analysis of HL60 differentiation (HL60diff)
	<ol>
		<li>Collect the cells and centrifuge the tubes at 300 g for 5 min at RT. Resuspend the pellet with complete Roswell Park Memorial Institute (RPMI) medium for counting using cell viability counter.</li>
		<li>To analyze the changes in cell-surface markers expression by flow cytometry. Place each 3 x 105&#160;undifferentiated HL60 cells (from cell culture) and 3 x 105&#160;HL60diff&#160;cells in 5 ml plastic tubes for Fluorescence-activated cell sorting (FACS) analysis and centrifuge the tubes at 300 g for 5 min at RT.</li>
		<li>Resuspend the pellets in 50 &#181;l Phosphate-buffered saline (PBS)-Fetal bovine serum (FBS) (4%). Label the cells with anti-human CD11b conjugated to AF700 or anti-human CD38 conjugated to APC (5 &#181;l/106&#160;cells). Incubate the cells in dark for 30 min at 4&#160;oC.</li>
		<li>Wash with 500 &#181;l PBS-FBS (4%) then centrifuge at 300 g for 5 min at RT. Resuspend the pellets in 200 &#181;l PBS-FBS (4%).</li>
		<li>Analyze on flow cytometer using the gating strategy of&#160;Figure 1A&#160;and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP); 633 nm laser: APC (660/20 BP), Alexa Fluor 700 (730/45 BP).</li>
		<li>To test the morphological changes in HL60diff, immobilize the cells onto glass slides for microscopic examination.
		<ol>
			<li>To do so, resuspend each 3 x 104&#160;undifferentiated HL60 cells (from cell culture) and 3 x 104&#160;HL60diff&#160;in 150 &#181;l complete RPMI medium. Assemble the glass slides, filter cards, and sample chambers in the cytospin centrifuge, ensuring that the centrifuge is balanced.</li>
			<li>Place each cell type into sample chamber and cyto-centrifuge at 1,500 rpm for 10 min at RT. Remove the slides, filter cards, and sample chambers from the centrifuge. Disassemble carefully, so as not to damage the cells on the slide. Discard filter cards and sample chambers.</li>
			<li>Stain the cells using Giemsa staining kit and keep the slides for 1 hr to dry. Examine the cells by microscope with 100X magnification.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>3-dimensional (3D) Culture 
	NOTE: Ensure that the materials used in this experiment are cold and the experiment is taking place on ice.
	<ol>
		<li>Resuspend each 5 x 104&#160;RL cells, either alone or mixed with HL60diff&#160;at ratio RL:HL60diff&#160;1:10, with 300 &#181;l basement membrane matrix using 1 ml pipette tip cut with a sterile scissor in order to widen the opening to about 2 to 3 mm. Avoid bubbles during this step.</li>
		<li>Seed each 300 &#181;l of cell suspension/well in 24-well plate. Avoid bubbles during this step. Incubate the plate for 30 min at 37 &#176;C with 5% CO2 then add 1 ml complete RPMI medium for each well and incubate for 7 days at 37 &#176;C with 5% CO2. Change the medium every two days. Add 10 nM vincristine at day 5.</li>
	</ol>
	</li>
	<li>FACS Analysis
	<ol>
		<li>After 7 days of culture, aspirate the medium and wash each well with 1 ml ice-cold PBS twice. Add 3 ml/well of ice-cold PBS-ethylenediaminetetraacetic acid (EDTA) (5 mM). Detach the gel from the bottom of the well by scraping using the bottom of 200 &#181;l pipette tip. Shake the plate gently on ice for 30 min.</li>
		<li>Transfer cell suspension into sterile 15 ml tube and shake the tubes gently on ice for another 30 min. Check for the appearance of homogeneous cell suspension. (If it&#39;s not the case, then shake the cells for longer time or add more PBS-EDTA).</li>
		<li>Centrifuge the tubes at 300 x g for 10 min at RT. Wash the pellets with PBS then centrifuge at 300 x g for 10 min at RT.</li>
		<li>Resuspend with PBS-FBS (4%) and label with anti-human CD19 antibody conjugated to PE-Cy7 and anti-human CD38 antibody conjugated to APC (5 &#181;l/106&#160;cells). Incubate in dark for 30 min at 4&#160;oC.</li>
		<li>Wash with 500 &#181;l PBS-FBS (4%) then centrifuge tubes at 300 g for 5 min at RT. Resuspend the cells with Annexin V and PI using commercial kit.</li>
		<li>Analyze on flow cytometer using the gating strategy of&#160;Figure 2A&#160;and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP), FITC (530/30 BP), PI (610/20 BP), PE-Cy7 (780/60 BP); 633 nm laser: APC (660/20 BP). Ensure the color compensation.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI 1640&#160;</td>
			<td>Gibco Invitrogen</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco Invitrogen</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS contains calcium and magnesium)</td>
			<td>Gibco Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Vincristine&#160;</td>
			<td>EG labo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel basement membrane matrix&#160;</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Put at 4 oC overnight before the day of the experiment</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexing V-FLOUS staining kit</td>
			<td>Roche</td>
			<td>11 988 549 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Kit RAL 555 Modified Giemsa staining kit</td>
			<td>Cosmos Biomedical</td>
			<td>CB361550-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>LSRII flow cytometry</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytocentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMR-XA microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4 Cell Viability Counter</td>
			<td>Nexcelom Bioscience</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Hirz, T., et al. <a href="https://app.jove.com/v/53846">Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents.</a> J. Vis. Exp. (2016).
This video demonstrates an assay investigating the role of neutrophil-like cells in safeguarding lymphoma cells against the cytotoxicity of chemotherapeutic agents. A 3D co-culture, consisting of lymphoma and neutrophil-like cells, is established and exposed to vincristine&#8212;a chemotherapy drug. Direct contact with the neutrophil-like cells induces an upregulation of anti-apoptotic proteins in the lymphoma cells, thereby promoting their survival in the presence of vincristine.

<img alt="Figure 1" src="/files/ftp_upload/22011/22011_Figure1.jpg" /> 
Figure 1.&#160;Structural Parameters of Differentiated HL60 Cells. (A)&#160;Forward-scatter (FSC) and side-scatter (SSC) plots represent HL60 and differentiated HL60 cells (HL60diff).&#160;(B)&#160;HL60 and HL60diff&#160;cells were labeled with anti-CD11b-AF700 or anti-CD38-APC followed by flow cytometry analysis. After gating on each cell popul...

1. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model

	Differentiation of Human ...

Take a multi-well plate containing 3D spheroid monocultures of lymphoma cells, cancerous white blood cells, and co-cultures of lymphoma and neutrophil-like cells in a basement membrane matrix.
Add a test drug that binds to intracellular tubulin and disrupts microtubule polymerization, resulting in cellular apoptosis.
In the co-culture, direct lymphoma cell contact activates the neutrophil-like cells, inducing cytokine secretion.
These cytokines interact with lymphoma cell receptors, triggering downstream signaling cascades and upregulating anti-apoptotic protein expression, promoting cell survival.
Discard the media. Add a dissociation buffer to disrupt cellular adhesion.
Scrape the spheroids and incubate them under agitation. Transfer the suspension to tubes and continue agitation to form single-cell suspensions.
Introduce a fluorophore-conjugated antibody cocktail that labels lymphoma and neutrophil-like cells.
Add fluorophore-tagged annexin V and propidium iodide to label apoptotic and dead cells.
Using flow cytometry, detect increased lymphoma cell survival in the co-culture, indicating neutrophil-mediated protection against the drug.

30 조회수. Determining the Role of Neutrophil-like cells in Lymphoma cell: test drug (림프종에서 호중구 유사 세포의 역할: 시험 약물에 대한 민감성)

비디오: Determining the Role of Neutrophil-like cells in Lymphoma cell: test drug (림프종에서 호중구 유사 세포의 역할: 시험 약물에 대한 민감성) - 실험

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

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Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

in vitro 종양 미세환경에서 면역 반응을 해부하기 위한 미세유체 공동 배양 모델

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Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

백혈구 탈출구 Murine 피부 및 종양 림프 혈관을 통해 측정

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug

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Determining the Role of Neutrophil-Like Cells in Lymphoma Cell Sensitivity to a Test Drug