This work was supported by Fondazione Istituto Italiano di Tecnologia. The authors would like to acknowledge Miltenyi Biotec GmbH (Bergisch Gladbach, Germany) for the sponsorship of this manuscript, Dr. Paolo Petrucciani (Department of Immuno-hematology and Transfusion Service, Hospital Lotti Pontedera, Pisa, Italy) for providing human buffy coats and Prof. Massimo Pasqualetti (Department of Biology - Unit of Cellular and Developmental Biology, University of Pisa, Pisa, Italy) for housing the mouse colony.

ETHICS&#160;STATEMENT
Human and animal samples were processed following the guidelines of the Italian Ministry of Health, the law 116/92 and the European Communities Council Directive 86/609/EEC.
1. Monocyte Cell Cultures
<ol>
	<li>Culture human THP-1 cells in T-75 flasks containing RPMI-1640 medium supplemented with 10% human serum, 50 U/ml penicillin and 50 mg/ml streptomycin, 0.05 mM &#946;-mercaptoethanol at 37 &#176;C in 5% CO2.</li>
	<li>Split cultures when confluent (every 3-5 days).</li>
</ol>
2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Buffy Coats
<ol>
	<li>Before starting the isolation protocol, prepare a solution of phosphate buffered saline (PBS) pH 7.2, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5% bovine serum albumin (BSA), adding 5 ml BSA Stock Solution to 95 ml Rinsing buffer (1:20 dilution). Degas the buffer and keep it cold (2-8 &#176;C). 
	IMPORTANT: Failure to degas the buffer may result in less than optimal results because bubbles may block the isolation column (see step&#160;3.2). 
	Note: Anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD) can be alternatively used. Conversely, other albumin proteins or sera from other species can replace BSA. Do not use buffers containing Ca2+ or Mg2+.</li>
	<li>Obtain buffy coats from healthy donors (aged 20-60 years old).</li>
	<li>Prepare 50 ml conical tubes for density gradient centrifugation. Determine the number of tubes required for processing the blood (each tube can process 35 ml of diluted blood) and add 15 ml of Ficoll-Paque to each empty tube.</li>
	<li>Dilute 8 ml of blood with 24 ml of PBS/BSA/EDTA solution (1:4 dilution).</li>
	<li>Carefully layer the diluted solution on top of Ficoll-Paque (density = 1.077 g/ml) in each of the 50 ml conical tubes. Do not mix the blood and Ficoll-Paque. 
	IMPORTANT: To avoid mixing of the blood and Ficoll-Paque, hold the tube at a 45&#176; angle and layer the blood mixture slowly.</li>
	<li>Centrifuge at 400 x g for 30 min at 4 &#176;C. Mononuclear cells (MNCs) will remain at the plasma-Ficoll-Paque interface, whereas granulocytes and erythrocytes sediment due to higher density at the osmotic pressure of Ficoll-Paque.</li>
	<li>Transfer the interphase cells (Peripheral blood mononuclear cells, PBMCs) to a new 50 ml tube filled with PBS/BSA/EDTA and centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Wash the pellet twice with PBS/BSA/EDTA to remove platelets. Spin at 200 x g for 10 min at 4 &#176;C.</li>
	<li>Culture the cells in complete RPMI-1640 with 10% human serum and antibiotics or proceed to monocytes purification.</li>
</ol>
3. Purification of Primary Monocytes from PBMCs
<ol>
	<li>Isolate monocytes from PBMCs by magnetic bead separation using Human Pan monocyte isolation kit:
	<ol>
		<li>Pass cells through a 30 &#181;m Nylon mesh (preseparation filters, 30 &#181;m) to remove possible cell clumps.</li>
		<li>Determine cell number using a hemocytometer.</li>
		<li>Centrifuge cell suspension at 300 x g for 10 min at room temperature (RT). Aspirate supernatant completely.</li>
		<li>Resuspend cell pellet in 30 &#181;l of PBS/EDTA/BSA buffer per 107 total cells.</li>
		<li>Add 10 &#181;l FcR Blocking Reagent per 107 total cells.</li>
		<li>Add 10 &#181;l of Biotin-Antibody Cocktail per 107 total cells.</li>
		<li>Mix well and incubate for 5 min at 2-8 &#176;C.</li>
		<li>Add 30 &#181;l of buffer per 107 total cells.</li>
		<li>Add 20 &#181;l of Anti-Biotin MicroBeads per 107 total cells.</li>
		<li>Mix well and incubate for 10 min at 2-8 &#176;C.</li>
		<li>Resuspend up to 108 cells in 500 &#181;l of PBS/EDTA/BSA buffer.</li>
	</ol>
	</li>
	<li>Magnetic separation
	<ol>
		<li>Accordingly to the total cell number, choose an appropriate MACS Column and MACS Separator (see MACS Column datasheet).</li>
		<li>Place the column in the magnetic field of the MACS Separator.</li>
		<li>Prepare column by rinsing with the appropriate amount of PBS/EDTA/BSA buffer (MS columns: 500 &#181;l; LS columns: 3 ml). 
		Note: Columns are &#34;flow stop&#34; and do not run dry .</li>
		<li>Apply cell suspension onto the column. Collect flow-through containing unlabeled cells, representing the enriched monocyte fraction.</li>
		<li>Wash column 3x&#160;with PBS/EDTA/BSA buffer (500 &#181;l for MS and 3 ml for LS per wash).</li>
		<li>Collect unlabeled cells that pass through, representing the enriched monocyte cells, and add to the flow-through from section 3.2.4. 
		Note: Wash the column by adding PBS/EDTA/BSA buffer aliquots only when the column reservoir is empty.</li>
		<li>(Optional) Remove column from the separator and place it on a suitable collection tube. Pipette the buffer onto the column (MS columns: 1 ml; LS columns: 5 ml). Immediately flush out the magnetically labeled nonmonocyte cells by firmly pushing the plunger into the column.</li>
		<li>Culture purified monocytes in complete RPMI-1640 with 10% human serum and antibiotics.</li>
	</ol>
	</li>
</ol>
4. Purification of Primary Monocytes from Whole Blood
<ol>
	<li>Purify monocytes from whole blood using whole blood monocyte isolation kit following manufacturer&#8217;s instructions.</li>
</ol>
5. Nanoparticle Internalization in Blood Leukocytes and Purified Monocytes
<ol>
	<li>Plate 5 x 105 cells/ml (1 ml/well) of the isolated PBMCs or the purified CD14 positive monocytes in a 12-well plate.</li>
	<li>Vortex FITC-SiO2 nanoparticle stock solution and resuspend in complete medium to a working concentration of 100 nM.</li>
	<li>Add 10 &#181;l of working nanoparticle suspension (100 nM) for treated samples to reach a nanoparticle final concentration of 1 nM. Add the same volume of complete medium for untreated controls to each well.</li>
	<li>After 1 hr internalization collect the samples in a 1.5 ml polypropylene tube and centrifuge them for 3 min at 6,000 x g at RT.</li>
	<li>Wash the pellet with 1 ml of running buffer 3 min at 6,000 x g at RT.</li>
	<li>Resuspend the pellet in 200 &#181;l of running buffer. The cells are now ready for reading by flow cytometry.</li>
</ol>
6. Isolation of Primary Mixed Glial Cultures (See Bertero&#160;et al.7)
7. Replating a Mixed Glial Confluent Cell Culture
<ol>
	<li>Wash a T-75 flask once with 10 ml of PBS (w/o Ca2+/Mg2+), then add 10 ml of Versene and incubate at 37 &#176;C for 5-7 min.</li>
	<li>Pipette the supernatant up and down several times in order to detach the cells from the T-75 surface and dissolve cell aggregates.</li>
	<li>Transfer the cell suspension in a 15 ml polypropylene tube. Collect a small amount of cellular suspension to count the cells with a hemocytometer.</li>
	<li>Centrifuge the cell suspension for 5 min at 300 x g at RT.</li>
	<li>Remove the supernatant and resuspend the pellet at a final concentration of 5 x 105 cells/ml in glial complete culture medium.</li>
	<li>Plate 0.5 ml/well in a 24-well plate and let the cells settle for 2-4 hr&#160;before nanoparticle treatment.</li>
</ol>
8. Nanoparticle Internalization in Microglia
<ol>
	<li>Add 500 &#181;l of complete glial culture medium (for untreated controls) or 500 &#181;l of a 2x&#160;Rhodamine-SiO2 nanoparticle suspension in complete glial culture medium (for treated samples) to each well.</li>
	<li>Pipette the cells to allow the detachment of nonrapid-adhering cells at the chosen time points and collect them in 2 ml polypropylene tubes.</li>
	<li>Add 500 &#181;l of Versene to each well and incubate the plate at 37 &#176;C for 5 min, then detach the remaining cell fraction and collect each sample with the corresponding nonadhering cells of the previous step.</li>
	<li>Centrifuge the samples for 3 min at 300 x g at RT.</li>
	<li>Resuspend the pellet in 100 &#181;l of AutoMACS Running buffer.</li>
</ol>
9. Staining with CD11b-VioBlue
<ol>
	<li>Add 10 &#181;l of VioBlue-CD11b human/mouse antibody to 100 &#181;l of cell suspension (1:11 ratio) in running buffer and incubate 10 min at 4 &#176;C.</li>
	<li>Add 1 ml of running buffer and mix the cell suspension.</li>
	<li>Centrifuge each sample for 3 min at 300 x g.</li>
	<li>Discard the supernatant and resuspend the pellet in 100-150 &#181;l of running buffer.</li>
	<li>Read the samples to the cytometer (store the samples at 4 &#176;C between the last two steps). 
	IMPORTANT: before analyzing the samples, proceed to compensation of overlapping signals in emission spectra observed between different fluorochromes (fluorochrome-conjugated nanoparticles or antibodies).</li>
</ol>
10. Spectral Spill Over Compensation
<ol>
	<li>Open the &#34;Instrument settings&#34; box (present in all the instrument software programs). 
	Note: If using a different instrument from the one reported in the Material Table, please refer to the instrument specific manual for the acquisition details.</li>
	<li>Acquire untreated sample (without nanoparticles) at low fluidic speed (if allowed by the instrument). During the acquisition, manually adjust forward and side scattering (FSC and SSC, respectively) by regulating the voltage channels. 
	Note: Modify the SSC and FSC axis scales in order to best visualize the complete cell population in the plot. An instrument setting template for each cell type of interest can be created, saved and used for future acquisitions.</li>
	<li>Open the specific &#34;region&#34; tab in the software. Draw an appropriate large region (&#34;gate&#34;) around the desired population. 
	Note: Internalization of nanoparticles induces a SSC shift of the cells. If the gate is too closely restricted around the cell population of interest, the acquired data will probably miss part of the cells to be detected.</li>
	<li>Use two unstained samples, one for use as blank sample and one for compensation against PI staining (optional), one single-stained sample with the appropriate fluorochrome for each fluorescence channel to be compensated. 
	Note: Use a different 1.5 ml sample tube for each fluorochrome-conjugated antibody to be used in the experiment labeling.</li>
	<li>Open the &#34;Compensation tab&#34; in the &#34;Instrument settings&#34; box. Increase or decrease the compensation factor during the acquisition until the fluorescence intensity in the spillover channel is roughly the same for the fluorochrome positive and negative population.</li>
	<li>Acquire nanoparticle-treated samples after the compensation has been adjusted.</li>
</ol>

<ol>
	<li>Zhang, X. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+nanomaterials+and+biological+systems:+Implications+to+personalized+nanomedicine.">Interactions of nanomaterials and biological systems: Implications to personalized nanomedicine.</a> Adv. Drug Deliv. Rev. 64, 1363-1384 (2012).</li><li>Radad, K., Al-Shraim, M., Moldzio, R., Rausch, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+benefits+and+hazards+of+engineered+nanoparticles.">Recent advances in benefits and hazards of engineered nanoparticles.</a> Environ. Toxicol. Pharmacol. 34, 661-672 (2012).</li><li>Dobrovolskaia, M. A., McNeil, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+properties+of+engineered+nanomaterials.">Immunological properties of engineered nanomaterials.</a> Nat. Nanotechnol. 2, 469-478 (2007).</li><li>Shi, C., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+recruitment+during+infection+and+inflammation.">Monocyte recruitment during infection and inflammation.</a> Nat. Rev. Immunol. 11, 762-774 (2011).</li><li>Sica, A., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+plasticity+and+polarization:+in+vivo+veritas.">Macrophage plasticity and polarization: in vivo veritas.</a> J. Clin. Invest. 122, 787-795 (2012).</li><li>Hubbell, J. A., Thomas, S. N., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+engineering+for+immunomodulation.">Materials engineering for immunomodulation.</a> Nature. 462, 449-460 (2009).</li><li>Bertero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+functionalisation+regulates+polyamidoamine+dendrimer+toxicity+on+blood-brain+barrier+cells+and+the+modulation+of+key+inflammatory+receptors+on+microglia.">Surface functionalisation regulates polyamidoamine dendrimer toxicity on blood-brain barrier cells and the modulation of key inflammatory receptors on microglia.</a> Nanotoxicology. 8, 158-168 (2013).</li><li>Kroll, A., Pillukat, M. H., Hahn, D., Schnekenburger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+engineered+nanoparticles+with+in+vitro+toxicity+assays.">Interference of engineered nanoparticles with in vitro toxicity assays.</a> Arch. Toxicol. 86, 1123-1136 (2012).</li><li>Malvindi, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SiO2+nanoparticles+biocompatibility+and+their+potential+for+gene+delivery+and+silencing.">SiO2 nanoparticles biocompatibility and their potential for gene delivery and silencing.</a> Nanoscale. 4, 486-495 (2012).</li><li>Bardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SiO2+NPs:+Promising+Candidates+for+Drug+and+Gene+Delivery.">SiO2 NPs: Promising Candidates for Drug and Gene Delivery.</a> Drug Deliv. Lett. 1, 9-12 (2011).</li><li>Bardi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+amino+functionalized+CdSe/ZnS+quantum-dot-Doped+SiO2+nanoparticles+with+primary+neural+cells+and+their+gene+carrying+performance.">The biocompatibility of amino functionalized CdSe/ZnS quantum-dot-Doped SiO2 nanoparticles with primary neural cells and their gene carrying performance.</a> Biomaterials. 31, 6555-6566 (2010).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+andgreen+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion andgreen fluorescent protein reporter gene insertion.</a> Mol. Cell Biol. 20, 4106-4114 (2000).</li></ol>

The authors declare that Miltenyi Biotech GmbH has sponsored the submission of this manuscript. HiQ-Nano Company (<a href="http://www.hiq-nano.com/index.html">http://www.hiq-nano.com/index.html</a>) is a nanotechnology spin off company arisen from IIT.

The experimental protocol presents very crucial points to be taken into account. It is really important to work at 4 &#176;C (on ice) and possibly in the dark during all the staining steps, because higher temperatures and lights may negatively affect the staining yield. Nanoparticles could be sonicated to be better resuspended just before use.
A correct flow cytometry analysis requires a correct calibration in the different channels. Calibration of the instrument should be performed before every experimental session. Besides technical issues with the instrumentation, there can be also problems with the antibody labeling. It is mandatory to use the antibody in an appropriate concentration. If the concentration is too high or too low, dissatisfying signal intensities can be the consequence.
The disadvantages of this technique concern the need of working with monodisperse samples, the inability to localize the site of origin of the signal (i.e. different cellular compartments). There are also some limits in the choice of fluorochromes to be used in combination: the wavelength of the excitation and the emission bands must be sufficiently separated to allow their appropriate measurement. If the used antibody spectra overlap, a correct compensation is required.
Flow cytometry is a powerful method for cell analysis in presence or absence of nanoparticles. This technique permits a multiparametric study of a cell, a high number of events examined, rapidity of analysis (more than 1,000 cells/sec), reproducibility and statistical readings. Samples can be processed without losing cell viability.
By using fluorescently labeled nanoparticles it is possible to qualify and quantify their internalization in cell subpopulations, which are identified by specific markers exposed on the cell membrane. Cell parameters can change in presence of specific nanoparticles. Depending on the aim of the research, these variations can be used to identify a specific phenomenon, such as side scattering of cells that proportionally increases with the increasing nanoparticle internalization rate.
Nanoparticle-induced modifications of cell surface can also represent a limitation of the technique. For this reason, cell membrane receptors turnover must be always taken in consideration and possibly known in advance to precisely characterize the cell population of interest. Extreme impairments of membrane osmosis in nanoparticle overloaded samples can lead to cell death.
Nanomaterial dose-dependent toxicity should be empirically tested for each cell population. Clearly defined dead cells must be excluded from the fluorescence quantification. For instance, Annexin V/PI staining is one of the several methods usually used to detect both necrotic and apoptotic cells.
GFP-expressing primary cells are also a powerful tool to select a certain cell subpopulation and collect data without prelabeling. The combination with complementary fluorescent nanoparticles allows a very fast and precise quantification of cell-nanoparticle interaction. Drug or gene delivery is thought to be improved in the future by the application of nanoparticles able to release a specific drug load into selected tissues.
Employment of nanoparticles as specific delivery carriers and/or immune modulators for pharmaceutics requires the knowledge of the biological environment (i.e. through flow cytometry) to study cell-nanoparticle interactions.

Engineered nanomaterials are nowadays inspiring the interest of life scientists for potential applications to biomedicine1. A wide variety of inorganic and organic materials can be used to produce nanostructures with different shapes, physical and chemical features. Among these structures, engineered nanoparticles of spherical shape demonstrated great potential for diagnostic and translational medicine2. Their core and surface design are driven by a possible application and it implies a profound study of target cell responses following nanoparticle contact and interaction. Nanoparticles that are thought to be deliberately administered to human subjects will come in direct contact with several types of immune cells. Their responsibility to maintain the body integrity makes them a crucial topic of investigation in nanomedicine3.
The cellular part of the innate immune system is mainly represented by phagocytes. Among them, the monocyte/macrophage lineage derived cells, including the central nervous system resident microglia, play a key role in the immune defense4,5. They are able to trigger protective responses within few hours after encountering with foreign bodies. Moreover, the monocytic cells coordinate and instruct the adaptive immune response through the release of cytokines. All these events occur even in presence of engineered materials, which are largely perceived as &#34;nonself&#34; by the immune system6.
Among the methods historically used in immunology for cell analysis, flow cytometry represents one of the most powerful tools. Furthermore, the availability of technologies for the identification or purification of a specific immune subpopulation (often exploiting the exclusion or the presence of one single membrane protein) allows a precise investigation of the effects of a certain nanoparticle on that particular primary cell type&#160;7. However, cells may present physiological and morphological alterations after exposure to nanoparticles. As well, nanoparticles may interfere with specific optical parameters, such as absorption or emission of light at defined wavelengths, influencing the obtained results8. So, limits of use and eventual adaptations of classical immunological assays to the study of novel materials should be considered.
This work concerns the detection of nanoparticle interactions with primary immune cells by flow cytometry. To address this issue, 50 nm FITC-SiO2 nanoparticles were employed as a model nanomaterial to describe the method. Silica particles can be produced in a very precise manner in the nano-metric scale. Size, shape, and surface properties, such as charge or hydrophobicity, can be finely tuned to increase their biocompatibility9. Many features of SiO2 nanoparticles allow them to be used as a model for drug delivery particles10. Furthermore, fluorescent dyes or quantum dots can be entrapped or linked to these particles offering useful nano-tools for imaging purposes11.

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">HBSS</td>
			<td style="width:107px;">Gibco</td>
			<td style="width:119px;">14170-088</td>
			<td style="width:256px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 (ATCC Modified)</td>
			<td>Gibco</td>
			<td>A10491-01</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, High Glucose, phenol red</td>
			<td>Gibco</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin, Liquid</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamycin</td>
			<td>Gibco</td>
			<td>15710-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horse Serum (lot n&#176; 1131917)</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-mercaptoethanol</td>
			<td style="width:107px;">Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin 2.5%</td>
			<td style="width:107px;">Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Pooled Serum</td>
			<td>Invitrogen</td>
			<td>34005100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versene</td>
			<td style="width:107px;">Invitrogen</td>
			<td>15040-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse I</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td>D5025-150KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11b-VioBlue human &#38; mouse</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-097-336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MACS BSA Stock Solution&#160;</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">autoMACS Rinsing Solution&#160;</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Running buffer</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">autoMACS Running Buffer</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Human Pan monocyte isolation kit&#160;</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-096-537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Whole Blood Column Kit</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Whole Blood CD14 MicroBeads, human</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-090-879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MS Column</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">LS Column</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MidiMACS Separator</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MiniMACS Separator</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Red Blood Cell Lysis Solution&#160;</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Pre-Separation Filters 30 &#181;m</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MACSQuant Analyzer flow cytometer</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MACSQuant Calibration Beads</td>
			<td style="width:107px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque Premium</td>
			<td style="width:107px;">GE Healthcare</td>
			<td style="width:119px;">GEH17544202</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:272px;">FITC-SiO2 nanoparticles (50nm, +45mV)</td>
			<td style="width:107px;">HiQ-Nano Company</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:272px;">Rhodamine-SiO2 nanoparticles (50nm, +45mV)</td>
			<td style="width:107px;">HiQ-Nano Company</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">12-well plate Falcon</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:119px;">353043</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of fluorescent nanoparticle interactions with primary immune cell subpopulations by flow cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Flow cytometry is a useful tool to identify and characterize different cells and it is the technique of choice to identify specific immune cells, such as monocytes, granulocytes, T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), and other subpopulations of leukocytes.
In the effort to better characterize white blood cells behavior in response to nanoparticles, we performed internalization assays with primary leukocytes isolated from the blood of healthy donors (Figures 1 and&#160;2) and with human monocyte&#160;cell line (THP-1 cells, Figure&#160;3).
As reported in Figure 1A, the three major blood leukocyte subpopulations were clearly identified by forward and side scattering after PBMCs isolation. Moreover, after FITC-SiO2 treatment, lymphocytes (grey), monocytes (blue) and granulocytes (red) have a different nanoparticle internalization rate as shown by green fluorescence intensity (Figure 1B). The described protocol allows the purification of primary CD14 positive monocytes from PBMCs. Figure 2A reports the flow cytometry dot plot of CD14+ monocytes in presence of FITC-SiO2. Figure 2B shows FITC-SiO2 internalization quantified in the same cells and expressed in logarithmic scale histogram.
Similar internalization experiments were performed on THP-1 monocytes treated with increasing concentrations of FITC-SiO2 nanoparticles. Untreated cells were used as negative control. Figure 3A shows a dose-dependent increase in side scattering with an unchanged forward scattering in THP-1 cell line. In Figure 3B, together with the histograms of SSC and FSC at each FITC-SiO2 nanoparticle concentration tested, mean fluorescence intensity (MFI) quantification is presented. This data suggest that treatment with FITC-SiO2 nanoparticles induces a dose-dependent internalization in monocytes highlighted by the enhancement of intracellular granularity (side scattering) and fluorescence (green channel).
To gain further insights into the type of interaction between immune cells and nanoparticles, primary mixed glial cultures were isolated and microglia, the central nervous system resident immune cells, was purified. The use of a transgenic mouse model expressing green fluorescent microglia permits the visualization of different neuro-inflammatory mechanisms. The transgenic mouse B6.129P-CX3CR1tm1Litt/J used in this work expresses the green fluorescent protein (GFP) under the control of CX3CR1 promoter12. After 7 days in vitro (DIV), fluorescence microscopy shows a mixed primary glial culture with a large number of astrocytes (GFP negative adherent cells) and some green cells (GFP positive, Figure 5A). In this mouse model, three glial subpopulations can be distinguished by flow cytometry with a single CD11b-antibody staining: the first CD11b-GFP- (astrocytes and other glial cells), a second distinct group of microglial CD11b+GFP+ cells, and a third CD11b+GFP- subpopulation (Figure 4A). These two latter subpopulations are both able to internalize nanoparticles with a slight increased efficiency by the GFP+ population (representing the patrolling immature microglia by the transcription of CX3CR1 promoter), as shown by flow cytometry analysis (Figure&#160;4B). The occurred internalization can be further verified by confocal microscopy using the same final concentration of Rhodamine-SiO2 nanoparticles as shown in Figure&#160;5B.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig1highres.jpg" src="/files/ftp_upload/51345/51345fig1.jpg" /> 
Figure 1. FITC-SiO2 nanoparticle internalization in isolated blood leukocytes. A) Representative forward scattering (FSC) versus side scattering (SSC) flow cytometry dot plot of Ficoll-Paque isolated blood leukocytes. B) Green fluorescence overlay histogram plot of the three major blood leukocyte cell subpopulations in presence of 1 nM FITC-SiO2 nanoparticles (+45 mV) for 1 hr. <a href="https://www.jove.com/files/ftp_upload/51345/51345fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig2highres.jpg" src="/files/ftp_upload/51345/51345fig2.jpg" /> 
Figure 2. FITC-SiO2 nanoparticle internalization in CD14+ purified monocytes. A) Representative forward scattering (FSC) versus side scattering (SSC) flow cytometry dot plot of purified CD14 positive monocytes. B) Green fluorescence histogram plot of the purified monocyte subpopulation in presence of 1nM FITC-SiO2 nanoparticles (+45 mV) for 1 hr. <a href="https://www.jove.com/files/ftp_upload/51345/51345fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig3highres.jpg" src="/files/ftp_upload/51345/51345fig3.jpg" /> 
Figure 3. Effects of FITC-SiO2 nanoparticle internalization on THP-1 cells. A) Representative forward scattering (FSC) versus side scattering (SSC) flow cytometry dot plot of THP-1 monocyte cell line, following 1 hr exposure of FITC-SiO2 nanoparticles increasing concentration. B) Concentration-dependent variation of the side scattering (SSC), forward scattering (FSC) and green fluorescence in presence of FITC-SiO2 nanoparticles (+45 mV) for 1 hr. <a href="https://www.jove.com/files/ftp_upload/51345/51345fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig4highres.jpg" src="/files/ftp_upload/51345/51345fig4.jpg" /> 
Figure 4. Rhodamine-SiO2 nanoparticle internalization into primary microglia isolated from B6.129P-CX3CR1tm1Litt/J mice. A) Green Fluorescent protein (GFP) versus CD11b-VioBlue flow cytometry dot plot of primary mixed glia isolated from B6.129P-CX3CR1tm1Litt/J mice. CD11b+GFP+ and CD11b+GFP- populations are shown in the upper right and lower right quadrants, respectively. B) Red fluorescence overlay histogram plot of microglia subpopulations in presence of 1 nM Rhodamine-SiO2 nanoparticles (+45 mV) for 30 min (red histogram) versus control (grey histogram). <a href="https://www.jove.com/files/ftp_upload/51345/51345fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51345/51345fig5highres.jpg" src="/files/ftp_upload/51345/51345fig5.jpg" /> 
Figure 5. Visualization of GFP+-microglia. (A) Fluorescence microscopy at 7 DIV and (B) Confocal microscopy of Rhodamine-SiO2 nanoparticle internalization (red arrows) in GFP+-microglia.<a href="https://www.jove.com/files/ftp_upload/51345/51345fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry at JoVE.com

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

detection-fluorescent-nanoparticle-interactions-with-primary-immune

Research

JoVE Journal

Immunology and Infection

17.4K Views.  Istituto Italiano di Tecnologia.  Анализ наночастиц взаимодействии с определенными субпопуляций иммунных клеток методом проточной цитометрии. 

Video: Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...