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 μm Nylon)</li>
 <li>Collection tubes: Two types of collection tubes can be used a) 12 x 75 mm polystyrene tubes (containing 300 μl 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 FcγR 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 °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 TCRβ and Alexafluor-700-conjugated anti-mouse CD4 to identify B cells (B220+TCRβ-) and CD4 T cells (CD4+TCRβ+). 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="Flow cytometry results; B cell, CD4 T cell purity diagrams; pre/post sort, TCRβ, CD4, B220 markers." data-altupdatedat="1747911167000" data-alt="Figure 1"> Figure 1. Purity of splenic B cells and CD4 T cells after FACS. Mouse splenocytes were stained with anti-mouse B220, TCRβ and CD4 antibodies. FACS for B cells was performed using a BD FACS Aria by gating on B220+TCRβ- 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 TCRβ staining on the y-axis. FACS for CD4 T cells was performed using the same flow cytometer by gating on TCRβ+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). TCRβ 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/pubmed?term=((Monoclonal+antibodies+and+the+FACS%3A+complementary+tools+for+immunobiology+and+medicine%5BTitle%5D)+AND+%22Immunol+Today%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGivan%2C+AL+%22Flow+cytometry+first+principles%22">Flow cytometry first principles</a>. , Wiley-Liss. New York. 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/pubmed?term=((Fluorescence-activated+single+cell+sorting+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Cloning+Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((Single-cell+genomics%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Safe+sorting+of+GFP-transduced+live+cells+for+subsequent+culture+using+a+modified+FACS+vantage%5BTitle%5D)+AND+%22Cytometry%22%5BJournal%5D)">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. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDiamond%2C+RA+%22In+living+color%3A+Protocols+in+flow+cytometry+and+cell+sorting%22">In living color: Protocols in flow cytometry and cell sorting</a>. DeMaggio, S. , Springer. Berlin. 800-800 (2000).</li>
<li>Karabinus, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+sorting+of+human+sperm%3A+MicroSort+clinical+trial+update%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">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/pubmed?term=((Seventeen-colour+flow+cytometry%3A+unravelling+the+immune+system%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">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/pubmed?term=((Interpreting+flow+cytometry+data%3A+a+guide+for+the+perplexed%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((Modern+flow+cytometry%3A+a+practical+approach%5BTitle%5D)+AND+%22Clin+Lab+Med%22%5BJournal%5D)">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

73.2K Views.  BloodCenter of Wisconsin. Voor veel wetenschappelijke studies die een biologische en chemische analyse van celpopulaties de cellen moeten worden in een hoge staat van zuiverheid. Fluorescentie geactiveerde cel sortering (FACS) is een superieure methode om zuivere celpopulaties te verkrijgen.

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

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Biology

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Neuroscience

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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 ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Fluorescence-Activated Single-Cell Sorting of Human Mesenchymal Stem Cells

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.
This warrants methodologies to provide purified, homogeneous populations of MSCs&#160;for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify&#160;stem cells from&#160;human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.
The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

Author Spotlight: Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and ...

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

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