We would like to thank Dr. Bill Cashdollar for his support in the use of the Flow Cytometry Core.

<ol><li>Before starting the process, the following items need to be to collected and prepared:
<ol style="list-style-type:lower-roman;">
 <li>Ice</li>
 <li>15 ml conical tubes for staining cells and 12 x 75 mm flow tubes for staining single color controls.</li>
 <li>Staining buffer: phosphate buffered saline (PBS) + 3% fetal calf serum (FCS)</li>
 <li>Pipettes</li>
 <li>Fc block (if required) and staining antibodies. Antibodies need to be titrated for optimal staining.</li>
 <li>Compensation beads</li>
 <li>Suspension buffer: Hank's Balanced Salt Solution (HBSS) + 25 mM HEPES + 3% FCS</li>
 <li>Trypan blue and hemocytometer</li>
 <li>Cell strainer (40 &mu;m Nylon)</li>
 <li>Collection tubes: Two types of collection tubes can be used a) 12 x 75 mm polystyrene tubes (containing 300 &mu;l FCS and 25 mM HEPES) or b) 15 ml conical tubes (containing 1 ml FCS + 25 mM HEPES). Any rich medium with high serum concentration can be used for the collection of sorted cells. </li>
</ol></li>
<li>Generate a single cell suspension of the starting cell population.</li>
<li>Optional: Enrich for the desired cell population by a bulk purification method such as complement depletion or magnetic sorting. The main advantage of the enrichment step is that it reduces the sort time.</li>
<li>Wash cells once with staining buffer.</li>
<li>Discard the supernatant and resuspend the cells in staining buffer at a concentration up to 50 x 106 for efficient staining.</li>
<li>Optional: For cells expressing high levels of FcR, block the receptors using an appropriate blocking method. A method of choice is the use of a mAb that binds to Fc&gamma;R on ice for 10-15 min. These antibodies are commercially available.</li>
<li>Add the appropriate mAb (at a predetermined concentration) to stain the desired cell population and incubate for 20-30 min on ice in the dark, followed by two washes with staining buffer. Optional: Staining with a vital dye, such as propidium iodide, can be included to discern dead cells (5). If multi-color staining is to be used, single color controls are required. We use BD CompBeads to prepare single color controls using the manufacturer's protocol.</li>
<li>If not using directly conjugated antibodies, repeat step 7 using the appropriate secondary antibody or Streptavidin conjugate.</li>
<li>After washing, resuspend the cells in culture medium and determine the cell concentration using a vital dye such as Trypan blue.</li>
<li>Adjust the cell concentration to 15-20 x 106/ml. For cell populations that form clusters, which can clog the instrument during sorting, filter the cells through a strainer.</li>
<li>Set up and optimize the cell sorter. The process of setting up a flow cytometer is varied depending on the manufacture and needs to be performed by appropriately trained personnel. The general recommendations are as follows:
<ol style="list-style-type:lower-roman;">
 <li>Select the appropriate nozzle depending on the cell type to be sorted.</li>
 <li>For sterile sorts, sterilize the instrument.</li>
 <li>Perform instrument quality control with beads to verify lasers are functioning, and it is sorting accurately. </li>
 <li>Install the required collection device and set up the side streams.</li>
 <li>Look at the instrument's laser and detector set up to determine the fluorescent labels that can be used (our BD FACS Aria has three lasers and can detect up to nine colors).</li>
 <li>Once it is determined what fluorescent labels will be used on the cell sorter, compensation can be performed (as explained below).</li>
</ol></li>
<li>Perform compensation using the negative control sample and the single positive controls. Compensation is necessary to remove the spectrum overlap between two detectors. It is pertinent to note that compensation is not fool proof and is adversely affected by how brightly certain markers stain, and by the fluorescence of cells that have low autofluorescence which can lead to poor resolution between dim and negative populations. 
 For best results, the experimental design should include using bright fluorochromes with markers that have low expression or that don't have a known staining pattern. Markers that provide good separation between cell populations and are highly expressed should be used with dimer fluorochromes such as ones excited by red or violet lasers.
<ol style="list-style-type:lower-roman;">
 <li>Compensation can be performed with compensation beads, which are polystyrene microparticles that have been coupled to an antibody specific for the Kappa light chain of Ig from mouse, rat, or rat/hamster. The beads are easy to stain, have a robust signal and provide an easy way of preparing single stained controls that have the same fluorophore as the experimental samples.</li>
 <li>Set up a template that includes a bivariate plot to display forward scatter (FSC) and side scatter (SSC), and one histogram for each fluorochrome that will be used. </li>
 <li>Run the negative control tube and adjust the forward scatter (FSC) and side scatter (SSC) to place the population of interest on scale.</li>
 <li>Adjust the fluorescence PMT settings to place the negative population or autofluorescence in the far left hand portion of the histogram.</li>
 <li>Record the negative control tube.</li>
 <li>Run the single positive control tubes and record the data for each tube. </li>
 <li>Draw an interval gate around the positive portion of the data on the histogram, and another interval gate on the negative portion of the histogram.</li>
 <li>Manual compensation is done by adjusting the median (median is a better estimate of central tendency than the mean on a logarithmic scale) of the positives until it is equal to the median of the negatives. This must be done for each of the fluorochrome labels used in the experiment. </li>
 <li>To adjust the median, adjust the spectral overlap values either higher or lower until the medians for each fluorescent parameter match as closely as possible.</li>
 <li>Compensation can also be done automatically with analysis software. Automatic compensation uses the recorded data from compensation controls to calculate an algebraic matrix for all the fluorophores used in the experiment. These values are used to subtract out the contributions of the non-primary colors that are bleeding into a primary color's detector. </li>
</ol></li>
<li>Record the experimental sample to be sorted and use gating tools and subsetting methods to define the populations of interest.
<ol style="list-style-type:lower-roman;">
 <li>Set up the experimental template with a dot plot that displays FSC vs. SSC and use a gating tool, polygon, rectangle, interval or quadrant to gate the population of interest. The scatter plot displays the physical properties of the cell which is distinct to the cell type. </li>
 <li>The best way to determine the fluorescence gating strategy is to use fluorescence minus one (FMO) controls. In a FMO control tube all reagents that are used in the experiment are included except for the one of interest. The FMO helps discriminate between dimly stained populations and broad negative populations. </li>
 <li>Use the data recorded from FMO tubes to determine where to place gates within the experimental tube. </li>
</ol></li>
<li>Once gates have been determined, the gates can be selected for sorting into external collection tubes. Up to four gates (or populations) can be sorted at one time depending on the instrumentation available.</li>
<li>Run the experimental sample tube at 4 &deg;C, turn on deflection plates, and sort the sample. 5 x 105 -1.5 x 106 cells can be sorted into a 12 x 75 mm tube and 1.5 x 106 - 4.5 x 106 cells can be sorted into a 15 ml conical.</li>
<li>Once the required number of cells has been obtained, manually stop sorting.</li>
<li>Perform a post-sort analysis to determine the purity of the sorted cell populations.</li>
</ol>
Representative results
We have shown here results for mouse splenic B and CD4 T cell sorting (Figure 1). Splenocytes were stained with PE Texas Red-conjugated anti-mouse B220, FITC-conjugated anti-mouse TCR&beta; and Alexafluor-700-conjugated anti-mouse CD4 to identify B cells (B220+TCR&beta;-) and CD4 T cells (CD4+TCR&beta;+). The sorting was performed with BD FACS Aria instrument. After sorting the B cell purity was &gt;97% and the CD4 T cell purity was &gt;98%.
<img src="/files/ftp_upload/1546/1546fig1.jpg" alt="Figure 1" /> Figure 1. Purity of splenic B cells and CD4 T cells after FACS. Mouse splenocytes were stained with anti-mouse B220, TCR&beta; and CD4 antibodies. FACS for B cells was performed using a BD FACS Aria by gating on B220+TCR&beta;- cells (Fig. 1A, left panel, lower right quadrant). A post sort analysis was performed to determine the purity of the B cells (Fig. 1A, right panel). B220 staining is shown on the x-axis and TCR&beta; staining on the y-axis. FACS for CD4 T cells was performed using the same flow cytometer by gating on TCR&beta;+CD4+ cells (Fig. 1B, left panel, upper right quadrant). A post sort analysis was performed to determine the purity of the CD4 T cells (Fig. 1B, right panel). TCR&beta; staining is shown on the x-axis and CD4 staining on the y-axis. The percent positive cells in each quadrant is shown.

<ol>
	<li>Herzenberg, L. A., De Rosa, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+and+the+FACS:+complementary+tools+for+immunobiology+and+medicine.">Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine.</a> Immunol Today. 21, 383-390 (2000).</li><li>Givan, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flow cytometry first principles. , 273-273 (2001).</li><li>Hewitt, Z., Forsyth, N. R., Waterfall, M., Wojtacha, D., Thomson, A. J., McWhir, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+single+cell+sorting+of+human+embryonic+stem+cells.">Fluorescence-activated single cell sorting of human embryonic stem cells.</a> Cloning Stem Cells. 8, 225-234 (2006).</li><li>Walker, A., Parkhill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomics.">Single-cell genomics.</a> Nat Rev Microbiol. 6, 176-177 (2008).</li><li>Sorensen, T. U., Gram, G. J., Nielsen, S. D., Hansen, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safe+sorting+of+GFP-transduced+live+cells+for+subsequent+culture+using+a+modified+FACS+vantage.">Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage.</a> Cytometry. 37, 284-290 (1999).</li><li>Diamond, R. A., DeMaggio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In living color: Protocols in flow cytometry and cell sorting. , 800-800 (2000).</li><li>Karabinus, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+sorting+of+human+sperm:+MicroSort+clinical+trial+update.">Flow cytometric sorting of human sperm: MicroSort clinical trial update.</a> Theriogenology. 71, 74-79 (2009).</li><li>Perfetto, S. P., Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seventeen-colour+flow+cytometry:+unravelling+the+immune+system.">Seventeen-colour flow cytometry: unravelling the immune system.</a> Nat Rev Immunol. 4, 648-655 (2004).</li><li>Herzenberg, L. A., Tung, J., Moore, W. A., Parks, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+flow+cytometry+data:+a+guide+for+the+perplexed.">Interpreting flow cytometry data: a guide for the perplexed.</a> Nat Immunol. 7, 681-685 (2006).</li><li>Tung, J. W., Heydari, K., Tirouvanziam, R., Sahaf, B., Parks, D. R., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+flow+cytometry:+a+practical+approach.">Modern flow cytometry: a practical approach.</a> Clin Lab Med. 27, 453-468 (2007).</li></ol>

FACS is a highly sophisticated technique for purifying cell populations of interest, in which a very high purity (95-100%) of the sorted population can be obtained. Therefore, this technique is particularly important for experiments, where high purity is an essential requirement (e.g. microarray analysis). FACS is particularly advantageous over other available methods of purification when a cell population needs to be purified based on a weekly expressed surface marker or when two or more populations need to be purified which have different levels of expression of the same surface marker. For example, the purification of marginal zone B cells (B220+CD21hiCD23int/low) and follicular B cells (B220+CD21int/lowCD23hi) based on expression levels of CD21 and CD23. Another growing application of FACS is single cell sorting allowing the analysis of individual cells (3, 4). FACS is also commonly used to sort cells expressing fluorescent proteins that are genetically expressed, such as green fluorescent protein tagged proteins (5). If a sterile sort is performed the cells can be cultured. However, cell viability and yield can be compromised during sorting. Viability can be improved by using a cell-dependent optimal temperature for the sorting and by processing collection tubes immediately after they reach capacity. Recovery can be increased by using polypropylene collection tubes, collecting sorted cells in serum rich media and inverting collection tubes intermittently, maintaining collection tubes at an optimal temperature and centrifuging sorted cells for ~10 min (http://cyto.mednet.ucla.edu/Protocol.htm).
The development of ultra high-speed sorters has extended the possibility of the application of flow sorting in clinical settings. The potential clinical applications of FACS include purification of blood stem cells from human blood for therapeutic purposes, applications in cancer therapy, amniocentesis replacement, sorting human sperm and early disease detections (6, 7).
For multicolor staining, antibody selection is a critical step and fluorochromes are chosen based on the detection system for each particular instrument. There are a number of companies that sell cell sorters and the laser configuration varies according to user needs. Thus one cannot simply use the exact same antibodies used in published studies without confirming compatibility. For optimal antibody selection, one should consider the expression level of the protein being examined. In general, the brightest available fluorochrome-tagged antibodies should be used to stain dimly expressed surface markers, while dim fluorochromes can be used for the staining of highly expressed surface markers (8). In addition, antibodies should be selected in such a way that overlap among their emission spectra is minimal. For multicolor staining, it is extremely difficult to choose fluorochrome that have no spectral overlap. In this situation, compensation is performed.
Compensation is necessary to mathematically eliminate the fluorescence overlap between the emissions spectrum of the various fluorochromes that is measureable by the detectors (8-10). In other words, the overlap fluorescence from one fluorochrome is subtracted from the emissions spectrum of another fluorochrome. The compensation calculation requires an unstained control and single positive controls for each fluorochrome used for a multicolor staining (9, 10). Compensation can be conducted using cells or beads. The beads are easy to stain, have a robust signal and provide an easy way of preparing single stained controls that have the same fluorophores as the experimental samples. Compensation beads are particularly advantageous when the cell surface protein of interest is expressed at a low level and when the cell population of interest is limiting in the initial starting cell population.
Once the antibodies have been chosen, their activity should be optimized by performing a titration analysis. This is particularly important if the FACS machine has highly powerful lasers. The use of suboptimal concentrations of staining antibodies can result in poor separation of the desired cell population from the total cell population. The use of high antibody concentrations increases the chance for antigen non-specific staining. Therefore antibody titration will allow the selection of the antibody concentration that gives the maximum brightness of the positive population and minimal background staining (8).
FACS is now a standard technique for the purification of subpopulations of cells. It can be used to separate any cell type in which a single cell suspension can be generated and antibodies are available to identify the desired cell population. FACS is the method of choice when highly pure cell populations are required.

purification of specific cell population by fluorescence activated cell sorting (facs)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Watch this Scientific Journal Video about Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS at JoVE.com

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

purification-specific-cell-population-fluorescence-activated-cell

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

72.9K Views.  BloodCenter of Wisconsin. For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations. 

Video: Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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 ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

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) ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...