flow cytometry-based analysis of dendritic cell activation using immune complexes

Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes

First, fix the cells in 1.8% buffered paraformaldehyde for 10 minutes at room temperature. Seed the cell suspension in each well of a U-shaped 96-well plate. Next, add varying dilutions of tumor-binding antibodies in each well. Thereafter, incubate the cells on ice for 15 to 20 minutes.
After incubation, wash the cells with 150 microliters of phosphate-buffered saline, then centrifuge at 400 times g for 5 to 10 minutes at 4 degrees Celsius. Post-centrifugation, expel the supernatant and wash the cells twice. Dissolve the cells in 100 microliters of FACS buffer, containing fluorophore-conjugated secondary antibody. Next, incubate the plate on ice for 15 to 20 minutes.
After 15 to 20 minutes, add 200 microliters of FACS buffer to wash the cells. Centrifuge the plate at 400 times g for 5 to 10 minutes. Decant the supernatant. Conduct flow cytometry to analyze the tumor binding, and to determine the minimal concentration of immunoglobulin G required to coat the cells.
Once the cells are coated with minimal immunoglobulin G concentration, add the CFSE-labeled tumor immune complex to the monocyte-derived dendritic cells in a 1:5 ratio. Then, incubate the mixture for 12 to 16 hours overnight in complete medium. Perform FACS analysis of the monocyte-derived dendritic cell activation.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Tumor-Associated Monocyte-Derived Dendritic Cells
<ol>
	<li>In a laminar flow hood, remove tumors from CO2&#160;euthanized mice and place tumors in RPMI medium without fetal bovine serum (FBS).&#160;&#160; 
	NOTE: Shaving the mice and spraying them with 70% ethanol before removing the tumors is highly recommended to minimize potential contaminations from the fur. Be sure that the tumor does not exceed 25 - 40 mm2&#160;since larger tumors have fewer dendritic cells (DC) that tend to be fragile and prone to undergo cell death. If larger numbers of DC are needed, each mouse can be injected with tumor cells at multiple sites.</li>
	<li>Under a sterile laminar hood, chop the tumors into small pieces (approximately 1 x 1 mm2) using surgery scissors.
	<ol>
		<li>Add all tumor fragments from one mouse into a 30-mL sterile flat-bottom tube with magnetic stir bars containing 5 mL of Hank&#39;s balanced salt solution (HBSS), 2 mg/mL collagenase IV, and 0.01 mg/mL DNase I. 
		CAUTION: Myeloid cells produce energy through glycosylation, even under a steady state. Therefore, only use media that contains glucose (e.g., HBSS, Roswell Park Memorial Institute (RPMI), and Dulbecco&#39;s Modified Eagle Medium (DMEM)), as glucose starvation for as little as 10 - 15 min will result in cell death and apoptosis within 24 h. Use only a low endotoxin collagenase IV, as collagenase is produced by Gram-positive bacteria (Clostridium histolyticum).</li>
	</ol>
	</li>
	<li>Stir at about 200 - 400 rpm in a 37&#160;oC incubator with a magnetic stirrer for 20-30 min.</li>
	<li>Add 5 mL of complete media and re-suspend vigorously.</li>
	<li>Filter cells through a 70-&#181;m cell strainer. Centrifuge at 4&#160;oC 400 rcf for 5 - 10 min to pellet the cells.&#160;&#160;&#160;&#160;&#160; 
	CAUTION: Do not attempt to isolate the cells directly following enzymatic digestion, as some tumors are necrotic and contain relatively large amounts of cell debris or extracellular matrix. Apply cells on a 15% density gradient medium to obtain only the cells.</li>
	<li>Suspend 9 mL of density gradient medium with 1 mL of 10x phosphate-buffered saline (PBS) to adjust osmolality and to obtain 100% Percoll stock solution. Mix 1.5 mL density gradient medium stock solution with 8.5 mL HBSS and mix it vigorously with tumor-derived cell pellet. Gently layer 2 mL of DMEM on the top of a 15% density gradient medium and centrifuge at 400 rcf for 20 min at room temperature. Discard the supernatants, as the cells will form a pellet at the bottom of the tube.
	<ol>
		<li>Wash the pelleted cells twice by re-suspending them in 10 mL HBSS containing 2% FBS, 1% penicillin/streptomycin, and 10 mM Ethylenediaminetetraacetic acid (EDTA) (Isolation Buffer), and centrifuge at 4&#160;oC 400 rcf for 5-10 min to pellet the cells.</li>
		<li>Count cells on a hemocytometer using trypan blue under a light microscope.</li>
	</ol>
	</li>
	<li>Re-suspend 1 x 108&#160;cells in 1 mL isolation buffer and incubate them at 4&#160;oC with 30 &#956;L of CD11b+&#160;conjugated magnetic beads for 15 min.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	CAUTION: Avoid the use of CD11c-conjugated beads, as this will activate the DC and induce cell death. Do not attempt to block Fc&#947;RII and Fc&#947;RIII with anti-CD16/32 antibodies. Blocking antibodies ligate Fc&#947;R, induce strong phosphorylation of mitogen-activated protein (MAP) kinases, and prime the DC.
	<ol>
		<li>Remove the excess unbound beads by adding 9 mL of isolation buffer and centrifuge cells at 400 rcf for 5 - 10 min at 4oC.</li>
	</ol>
	</li>
	<li>Aspirate the supernatant and re-suspend cells in 1 mL isolation buffer. Apply the cells on a prewashed magnetic column. Wash the column twice with 3 mL of isolation buffer.
	<ol>
		<li>Remove the column from the magnet. Pipette 6 mL of isolation buffer onto the column and flush out the magnetically labeled cells into a sterile collection tube by pushing the plunger. Centrifuge cells at 400 rcf for 5 - 10 min at 4&#160;oC.</li>
		<li>Re-suspend cells at 100 &#956;L per 1 x 107&#160;cells and stain with the following fluorophore-conjugated antibodies: Linage negative (TCRb, Siglec F, B220, CD19, FceRI&#160;and Ly6G),&#160;MHCII, Ly-6C.</li>
	</ol>
	</li>
	<li>Sort cells by gating the small cells, using side scatter (SSC) and forward scatter (FSC), and culture in a complete medium supplemented with 5 ng/mL GM-CSF&#8212;Incubate cells for 1 hour at 37&#160;oC to allow macrophages to adhere to the plate. Then, transfer the loose and non-adherent cells to a new culture dish. 
	NOTE: Mouse MoDC are defined as CD11b+/CD11c+/MHCIIhi/Ly6Clo/int. Expression of Ly6C may vary dramatically between tumor models, and in some models (e.g., LMP), MoDCs completely lack Ly6C expression. One 100 mm3&#160;B16F10 tumor typically contains 5 x 104&#160;of DC, resulting in approximately 4 - 5 x 106&#160;total cells. Of these cells, 10 - 15% are immune cells and 8 - 10% are MoDC. Increasing the DC numbers can be achieved by injecting mice with tumors at multiple sites. Sort only small SSC/FCS cells, as other myeloid cells can express markers such as CD11c and Ly6C.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	OPTIONAL: Sort the tumor-infiltrating monocyte as small SSC/FSC cells that express Ly6Chi&#160;and are negative for MHCII. Afterward, culture monocytes&#160;in vitro&#160;with 50 ng/mL GM-CSF to obtain DC.</li>
</ol>
2. Preparation of Tumor-IgG Immune Complexes
<ol>
	<li>Culture tumor cells in a 75 cm2&#160;culture flask to 70% confluence in complete DMEM media.
	<ol>
		<li>Add 2 mL of 0.25% trypsin/EDTA to detach cells from the culture flask and monitor cell morphology under a light microscope to avoid over-trypsinization.</li>
		<li>Add 8 mL of complete culture media (per 2 mL of trypsin) to inhibit trypsin digestion, and centrifuge at 4&#160;oC, 400 rcf for 5-10 min to pellet the cells. 
		NOTE: Check tumor cells for&#160;Mycoplasma&#160;using a commercial PCR kit. Test for Gram-negative bacteria and fungal endotoxins using&#160;Limulus Amebocyte Lysate&#160;(LAL) assay). Serum must be filtered through 0.22 &#956;M and tested for the 9 Code of Federal Regulations viruses. Additionally, test the culture media and serums by dropping 100 - 200 &#956;L onto LB agar or broth and culture for 2 days at 37&#160;oC.</li>
		<li>Wash cells again from trypsin and serum remains by re-suspending them in 10 mL PBS and centrifuge at 400 rcf for 5 - 10 min. Aspirate supernatant and repeat the wash 2 more times.</li>
	</ol>
	</li>
	<li>Fix cells in 1.8% buffered paraformaldehyde for 10 min at room temperature.
	<ol>
		<li>Wash the cells by re-suspending them in 10 mL PBS and centrifuge at 4&#160;oC 400 rcf for 5 - 10 min.</li>
		<li>Aspirate supernatants and repeat wash two more times. 
		Optional: For tumor uptake assays, cells can be labeled by incubating them for 5 min at 37 oC in PBS containing 1 &#956;M carboxyfluorescein succinimidyl ester (CFSE). CFSE is then quenched with complete media for 10 min in ice. The cells should be washed extensively in PBS containing 2% serum to remove residual dye.</li>
	</ol>
	</li>
	<li>Re-suspend cells in fluorescence-activated cell sorting (FACS) buffer (PBS supplemented with 2% FCS + 5 mM EDTA) and 0.5 &#956;g/mL of anti-CD16/32 to block potential non-specific protein-protein interactions. Plate in a U-shape 96 wells/plate at a concentration of 1 x 105&#160;cells per 100 &#956;L.
	<ol>
		<li>Add different dilutions of tumor-binding antibodies ranging from 5 &#956;g - 5 ng/1 x 105&#160;cells. Incubate the plate on ice for 15-20 min. 
		NOTE: IgG antibodies were isolated from the serum of na&#239;ve 20-24 weeks old female mice on protein A columns, as described.</li>
	</ol>
	</li>
	<li>Wash cells by adding 150 &#956;L of PBS and centrifuging the plate at 4&#160;oC 400 rcf for 5 - 10 min.
	<ol>
		<li>Discard the supernatants and repeat the wash twice. Re-suspend cells in 100 &#956;L FACS buffer containing fluorophore-conjugated secondary antibody. Incubate plate on ice for 20 min.</li>
		<li>Wash cells by adding 200 &#956;L of FACS buffer and centrifuging the plate at 400 rcf for 5 - 10 min. Discard the supernatants and repeat the wash.</li>
		<li>Analyze tumor binding by flow cytometry and determine the minimal concentration required to coat the cells.&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: Antibodies that do not increase mean fluorescence intensity (MFI) of stained tumor cells by at least fivefold over isotype control should not be used in subsequent functional assays.</li>
	</ol>
	</li>
</ol>
3. Activation of MoDC with Tumor-IgG IC
<ol>
	<li>Coat tumor cells with minimal IgG concentration, as described in sections 2.1 through 2.4.3
	<ol>
		<li>One day before activating MoDC with tumor IC, replace MoDC culture media containing GM-CSF. To do so, gently aspirate the media and wash the cells once with pre-warmed complete culture media. 
		NOTE: Isolated mature tumor-associated MoDC should be cultured for at least 2 - 3 h (or even overnight) after sorting in complete media without GM-CSF, and before activating them with IC.</li>
		<li>For tumor uptake analyses, add the CFSE-labeled tumor IC to MoDC at a ratio of 1:5 (IC: MoDC) and incubate overnight for 12 - 16 h in 1 mL of complete media per 1 x 106&#160;DC.</li>
		<li>For FACS analyses of MoDC activation experiments, add tumor-IC at a 1:1 (IC: MoDC) ratio and incubate overnight for 12 - 16 h. 
		NOTE: Including a positive control well is highly recommended, in which MoDC are stimulated with 1 &#956;g/mL of LPS or other TLR agonists.</li>
		<li>Following overnight activation, aspirate the supernatants and wash cells gently three times with isolation buffer or 10 mM EDTA HBSS.</li>
		<li>To detach DC from the plate, incubate cells for 2 - 3 min in 1 mL HBSS containing 10 mM EDTA and detach cells by vigorous pipetting.</li>
		<li>Centrifuge cells at 400 rcf and re-suspend 1 x 106&#160;DC in 90 &#956;L PBS supplemented with 2% FCS, 5 mM EDTA (FACS buffer) and 0.5 &#956;g of blocking antibodies. Incubate on ice for 5 - 10 min.</li>
		<li>Add 10 &#956;L of staining antibodies mixture to the cells and incubate on ice for 15 min.</li>
		<li>Add 2 mL FACS buffer to cells and centrifuge at 4&#160;oC 400 rcf for 5 - 10 min.</li>
	</ol>
	</li>
	<li>Re-suspend cells in 200 &#956;L FACS buffer.
	<ol>
		<li>Add 0.5 - 1 &#956;g/mL of DAPI 1 - 2 min before running the samples to exclude dead cells from analyses. Do not over-incubate DAPI, as MoDC will take it up within 10 - 15 min.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE-Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>StemCell Technologies</td>
			<td>7820</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19861</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C9697-50MG</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>PE-CD11c</td>
			<td>Biolegend</td>
			<td>117307</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD11b</td>
			<td>Biolegend</td>
			<td>101211</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 MHCII</td>
			<td>Biolegend</td>
			<td>107641</td>
			<td></td>
		</tr>
		<tr>
			<td>AF48- CD86</td>
			<td>Biolegend</td>
			<td>105017</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cy7-Ly-C6</td>
			<td>Biolegend</td>
			<td>108423</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7-CD15</td>
			<td>Biolegend</td>
			<td>135523</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Santana-Magal, N., et al. <a href="https://app.jove.com/v/57188">Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes.</a>&#160;J. Vis. Exp. (2018)
This video demonstrates a protocol for assessing the activation of dendritic cells using immunoglobulin G (IgG)-coated tumor cells. Dendritic cells internalize the IgG-coated tumor cells, process the tumor antigens, and present them on the surface through MHC-II molecules, along with the co-expression of the costimulatory molecule CD86. These dendritic cells then undergo MHC-II and CD86-targeted immunostaining and flow cytometry analysis to identify their activation state.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Tumor-Associated ...

Begin with a chemically fixed tumor cell suspension.
Add an optimum concentration of tumor-binding immunoglobulin G that binds to tumor cell surface antigens, forming immune complexes, or ICs.
Transfer the ICs to a culture plate containing adhered dendritic cells. Incubate.
The dendritic cell recognizes the IC, internalizes it, and processes it into peptide fragments.
The fragments are loaded onto major histocompatibility complex class II or MHC-II molecules and presented on the cell surface, along with the costimulatory molecule CD86.
Remove media. Add a cell dissociation buffer and pipette vigorously to detach the cells.
Transfer the cells to a tube.
Centrifuge and resuspend the cells in a blocking solution to block non-specific interactions.
Overlay with a fluorophore-labeled antibody cocktail targeting MHC-II and CD86.
Add a DNA-binding dye and incubate briefly to stain dead cell nuclei.
Using flow cytometry, identify the live cells co-expressing MHC-II and CD86, confirming IC-mediated dendritic cell activation.

39 Views. Oparta na cytometrii przepływowej analiza aktywacji komórek dendrytycznych przy użyciu kompleksów immunologicznych

Video: Oparta na cytometrii przepływowej analiza aktywacji komórek dendrytycznych przy użyciu kompleksów immunologicznych - Experiment

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

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The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

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Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes

Transkrypcja

Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes