We thank Aaron Rae (Emory University Department of Pediatrics and Children's Healthcare of Atlanta Flow Core) for cell sorting. This work was supported by NIH grant AA01787001, a Career Development Award from the Crohn's and Colitis Foundation of America, and an Emory-Egleston Children's Research Center seed grant to T.L.D.

1. Dissection and Dissociation of Intestinal Epithelial Cells
Preparation of reagents and equipment:
<ul>
	<li>Warm Ca2+/Mg2+-free PBS (CMF PBS) to room temperature.</li>
	<li>Warm Ca2+/Mg2+-free HBSS with 5% FBS (CMF HBSS/FBS) and 2mM EDTA to room temperature.</li>
	<li>Warm Orbital shaker to 37 &deg;C.</li>
</ul>
Note: Steps 1.1 to 1.7 must be performed as quickly as possible to minimize the extent of cell death and to achieve maximum cell yield.
<ol>
	<li>Euthanize mice in a CO2 chamber and spray 70% ethanol onto the abdomen and thorax.</li>
	<li>Make a small horizontal incision in the middle of the abdomen with a scissor and peel back the skin to expose the peritoneum.</li>
	<li>Proceed to separate the stomach from the upper small intestine by cutting at the pyloric sphincter. Tease away the mesentery with forceps and cut again at the ileo-cecal valve to free the entire small intestine from the large intestine. Make a cut at the anal verge and again tease apart the mesentery until the large intestine is free.</li>
	<li>Cut open the colon longitudinally using scissors and wash off fecal contents and mucus from the intestinal lumen in CMF PBS at room temperature.</li>
	<li>Use scissors and forceps to macroscopically dissect out the Peyer's patches along the anti-mesenteric surface of the small intestine and cut open the small intestine longitudinally.</li>
	<li>Wash the small intestine lumen of fecal contents and mucus in CMF PBS at room temperature.</li>
	<li>Separately cut the small/large intestine into approximately 1.5 cm pieces and place into separate 50 ml conical tubes containing 30 ml of pre-warmed CMF HBSS/FBS and 2mM EDTA. Do not add more than 1 intestine per 50 ml conical tube.</li>
	<li>Horizontally place each 50 ml conical tube into an orbital shaker and shake for at 250 rpm for 20 min at 37 &deg;C.</li>
	<li>Place a single mesh wire strainer over a waste bucket and pour the contents of each 50 ml conical tube through to recover the 1.5 cm pieces of intestine and place them in separate 50 ml conical tubes containing 30 ml of pre-warmed CHBSS/FBS with 2 mM EDTA.</li>
	<li>Repeat 1.8.</li>
</ol>
2. Tissue Digestion and Isolation of Intestinal Cells
Preparations of reagents and equipment:
<ul>
	<li>Collagenase solution: 1.5 mg/ml Type VIII Collagenase dissolved in pre-warmed CMF HBSS/FBS with 40 &mu;g/ml of DNase I.</li>
	<li>Pre-warmed CMF HBSS/FBS and 2mM EDTA.</li>
	<li>Pre-warmed orbital shaker at 37 &deg;C.</li>
	<li>Ice-cold CMF HBSS/FBS.</li>
</ul>
<ol>
	<li>After the second round of shaking, pour the contents of each 50 ml conical tube through the strainer and transfer 1.5 cm pieces of intestine to a small plastic weigh boat after dabbing away excess media using a paper towel.</li>
	<li>Rapidly mince the 1.5 cm pieces of intestine using scissors directly in the weight boat and add minced intestine to 20 ml of collagenase solution. Horizontally place each 50 ml conical tube into an orbital shaker and digest at 200 rpm for 10-20 min at 37 &deg;C. Please see discussion below regarding optimization.</li>
	<li>Briefly vortex to ensure thorough dissociation of any remaining intestinal tissue and filter through a 100 &mu;m cell strainer directly into a 50 ml conical tube.</li>
	<li>Top off each 50 ml conical tube with CMF HBSS/FBS and centrifuge at 1500 rpm for 5 min at 4 &deg;C. If a solid pellet is not observed for colon samples after centrifugation, the samples should be centrifuged again for 3.5 min. Repeat this wash step once more.</li>
	<li>Pour off the supernatant and resuspend the cell pellet in ice-cold CMF HBSS/FBS and place samples on ice.</li>
	<li>Proceed to Section 3 for FACS acquisition/analysis or Section 4 for magnetic-bead enrichment for high-speed cell sorting.</li>
</ol>
3. Antibody Staining for Multi-Color Flow Cytometric Analysis of DCs and Macrophages
Preparations of reagents and equipment:
<ul>
	<li>Ice-cold CMF PBS.</li>
	<li>Ice-cold staining buffer (CMF PBS + 5% FBS).</li>
	<li>Prepare dead cell stain in ice-cold CMF PBS at 1:1000 dilution using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit.</li>
	<li>Prepare the antibody staining cocktail by adding the following fluorescence-labeled monoclonal antibodies (mAbs) to the ice-cold staining buffer: CD45-PerCP, CD103-PE, CD11c-APC, MHC-II (I-Ab)-Alexa Fluor 700, CD11b-eFluor 450, F4/80-PE-Cy7.</li>
</ul>
<ol>
	<li>Transfer cells into a 5 ml polystyrene round-bottom (FACS) tube.</li>
	<li>Wash cells twice in ice-cold CMF PBS.</li>
	<li>Incubate samples with dead cell stain for 15 min on ice in the dark.</li>
	<li>Wash cells twice in ice-cold CMF PBS.</li>
	<li>Block cells with 2.4G2 anti-Fc&gamma;RIII/II in ice-cold staining buffer for 10 min on ice.</li>
	<li>Wash cells in ice-cold staining buffer.</li>
	<li>Incubate samples with antibody staining cocktail for 20 min on ice in the dark.</li>
	<li>Wash cells with ice-cold staining buffer twice and resuspend samples in 400 &mu;l of ice-cold staining buffer and pass through 40 &mu;m filter cap on FACS tubes.</li>
	<li>Acquire samples on LSR II cytometer (BD) as defined by gating strategy in Section 5 and Figure 1.</li>
</ol>
4. Enrichment of DCs and Macrophages from the Intestine
Preparations of reagents and equipment:
<ul>
	<li>Ice-cold staining buffer (CMF PBS + 5% FBS).</li>
</ul>
<ol>
	<li>Incubate single cell suspension obtained from step 2.5 with CD11b and CD11c MACS beads according to manufacturer's instructions.</li>
	<li>Wash cells with ice-cold staining buffer followed by centrifugation.</li>
	<li>Discard supernatant and resuspend the cell pellet in 1 mL ice-cold staining buffer and pass through a 100 &mu;m cell strainer followed by a 40 &mu;m cell strainer.</li>
	<li>Enrich for magnetic bead-attached cells by positive selection using MACS LS magnetic column.</li>
	<li>Repeat step 4.2 and discard supernatant.</li>
	<li>Incubate cells with surface marker mAbs as described in step 3.7.</li>
	<li>Wash magnetic bead-enriched cells twice with ice-cold staining buffer. Resuspend cell pellets in 500 &mu;L ice-cold staining buffer without sodium azide, and pass through 40 &mu;m cell strainer into a FACS tube.</li>
	<li>Proceed to FACS-sorting on the BD ARIA II Cell Sorter to sort intestinal DC and/or macrophage subsets of interest.</li>
</ol>
5. Gating Strategy for LP APCs
Note: Please note that unstained intestinal cells may be utilized as a negative control to assist in the proper placement of the gates to separate positive and negative populations.
<ol>
	<li>As shown in Figure 1, create a dot plot and exclude cells that are positive for the dead cell stain (Fig. 1A) followed by the exclusion of doublet events (Fig. 1B and C). Then, gate on the cells of interest according to forward and side scatter making sure to exclude debris (Fig. 1D).</li>
	<li>Create another dot plot and further gate on CD45+ and I-Ab+ cells, which phenotypically characterizes APCs (Fig. 1E).</li>
	<li>On a separate dot plot, analyze for CD11b and CD11c expression to distinguish specific DC and macrophage subsets (R1, R2, and R3; Fig. 1F). Cells of R1 are CD11c+ CD11bdull/- cells. The region, R2, is delineated such that these cells have similar levels of surface expression for CD11c as in cells of R1 but cells of R2 also express CD11b. Thereafter, the R3 region is designated for cells that are CD11b+ and CD11cdull/-.</li>
	<li>Analyze regions R1, R2, and R3 further for F4/80 and CD103 expression to differentiate macrophage and DC populations, respectively. The CD11c+ CD11bdull/- cells of R1 express high levels of the &alpha;E integrin, CD103, and low levels of F4/80 (Fig. 1G). CD11b+CD11c+ cells of R2 are composed of both DCs and macrophages based on their dichotomous expression of CD103 and F4/80 (Fig. 1H). Lastly, CD11b+CD11cdull/- cells of R3 constitute macrophages based on the phenotypic profile of F4/80+ and CD103- (Fig. 1I)16.</li>
</ol>
6. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/4040/4040fig1.jpg" /> 
Figure 1. Gating strategy for intestinal DCs and macrophages. Dead cells (A) and doublets (B and C) were first excluded from the analysis and then small intestinal cells were gated accordingly to forward and side scatter (D), and APCs were defined as CD45+I-Ab+(E). Macrophages and DCs were identified by the expression of CD11b and CD11c (F). CD103 and F4/80 expression for cells pre-gated on R1 (G), R2 (H) and R3 (I) populations was analyzed.
<img alt="Figure 2" src="/files/ftp_upload/4040/4040fig2.jpg" /> 
Figure 2. Cell yield and antibody staining quality depends on digestion time. CD11b and CD11c staining pattern and total cell yield of under- (A, D), optimally- (B, E) or over-digested (C, F) intestinal tissue.
Intestinal cells were isolated from a C57BL/6 mouse small intestine and DCs and macrophages were analyzed by FACS on the BD LSR II. Voltage and compensation were set using unstained and single fluorochrome-stained splenocytes. Dead cells (Fig. 1A) and doublets (Fig. 1B and C) were first excluded from the analysis. Cells of interest were then analyzed according to forward and side scatter (Fig. 1D) followed by gating on CD45+ and I-Ab+ cells (Fig. 1E). Thereafter, CD11b and CD11c expression was assessed among the CD45+I-Ab+ cells to delineate three regions (R1, R2, and R3; Fig. 1F). CD103 and F4/80 expression in the three regions was evaluated to distinguish between DCs and macrophages, respectively. The CD11c+ CD11bdull/- cells of R1 expressed high levels of the &alpha;E integrin, CD103, and low levels of F4/80 (Fig. 1G). CD11b+CD11c+ cells of R2 were composed of both DCs and macrophages based on their dichotomous expression of CD103 and F4/80 (Fig. 1H) while CD11b+CD11cdull/- cells of R3 constitute macrophages based on the phenotypic profile of F4/80+ and CD103- (Fig. 1I)16. Macrophages within the R2 gate and macrophages in the R3 gate have similar forward and side scatter properties and are distinguishable by CD11c expression. The functional dichotomy of these subsets remains incompletely understood.
The relationship between the duration of tissue digestion on total cell yield and the expression of CD11b and CD11c is illustrated in Figure 2. Intestinal tissue that was digested for 3 min (under-digestion) yielded low total cell number (Fig. 2D) and thus few DCs and macrophages available for characterization (Fig. 2A). Tissue digestion for 11 min produced a robust yield of live cells (Fig. 2E) with populations of DCs and macrophages that expressed high levels of CD11b and CD11c and were phenotypically distinct (Fig. 2C). In contrast, digestion for 50 min (over-digestion) resulted in a similar cell yield when compared to optimized digestion (Fig. 2E and F), however, delineation of different cell populations using CD11b and CD11c became more obscure as the expression of CD11c diminished (Fig. 2C) and the number of dead cells increased (data not shown).

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No conflicts of interest declared.

<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figure 3"/> 
Figure 3. Factors important for the optimization of cell yield and surface antigen expression. Cell yield and surface antigen expression are directly affected by the duration of tissue digestion, the specific characteristics of the collagenase, the degree of tissue mincing, and the presence or absence of inflammation, which may affect tissue integrity and cellularity. Prolonged tissue digestion may result in decreased cell viabili...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>1X PBS, Ca2+- and Mg2+-free</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hank's balanced salt solution (HBSS) with phenol red</td>
 <td>Fisher Scientific</td>
 <td>SH3001603</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>Sigma</td>
 <td>S6014</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1M HEPES in 0.85% NaCl</td>
 <td>Lonza</td>
 <td>17-737E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS)</td>
 <td>Atlanta biologicals</td>
 <td>S11150H</td>
 <td>Heat-inactivated</td>
 </tr>
 <tr>
 <td>0.5M EDTA (pH 8.0)</td>
 <td>Cellgro</td>
 <td>46-034-CI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase type VIII </td>
 <td>Sigma</td>
 <td>C2139</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td>14785000</td>
 <td>Stock solution: 100mg/ml</td>
 </tr>
 <tr>
 <td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for 405 nm excitation</td>
 <td>Invitrogen</td>
 <td>L34957</td>
 <td>Use at 1:1000</td>
 </tr>
 <tr>
 <td>CD45-PerCP mAb (30F11)</td>
 <td>BD</td>
 <td>557235</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>CD103-PE mAb (M290)</td>
 <td>BD</td>
 <td>557495</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>Fc&#x03B3;RIII/II mAb (2.4G2)</td>
 <td>BD</td>
 <td>553141</td>
 <td>Use at 1:200</td>
 </tr>
 <tr>
 <td>CD11c-APC mAb (N418)</td>
 <td>eBioscience</td>
 <td>17-0114-82</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>MHC-II (I-Ab)-Alexa Fluor 700 mAb </td>
 <td>eBioscience</td>
 <td>56-5321-82</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>CD11b-eFluor 450 mAb (M1/70)</td>
 <td>eBioscience</td>
 <td>48-0112-82</td>
 <td>Use at 1:200 </td>
 </tr>
 <tr>
 <td>F4/80-PE-Cy7 mAb (BM8)</td>
 <td>eBioscience</td>
 <td>25-4801-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11b microbeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-049-601</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11c microbeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-052-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL conical tubes</td>
 <td>BD Falcon</td>
 <td>352098</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Single mesh wire strainer</td>
 <td>Chefmate</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Small weigh boat</td>
 <td>Fisher Scientific</td>
 <td>08-732-116</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>40 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5 mL polystyrene round-bottom tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>MaxQ 4450 benchtop orbital shaker</td>
 <td>Thermo Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LS MACS column</td>
 <td>Miltenyi Biotec</td>
 <td>130-042-401</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LSR II</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FACSAria II</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

isolation-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

39.0K 次观看.  Emory University. 在这里，我们详细介绍了小鼠肠道树突状细胞（DCs）和巨噬细胞的快速分离方法。使用多色流式细胞仪分析细胞分选磁珠富集而被用来产生高纯度的功能研究人口肠道DC和巨噬细胞的表型特征。

视频: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.