Funding of this project was provided by the National Science Foundation grants OCE1029607 (to X.M.) and MCB0702125 (to M.A.M.) and the Gordon and Betty Moore Foundation (to M.A.M.).

1. Water sample processing
<ol>
 <li>Filter 10L environmental water through 1 &mu;m-pore-size membrane filters to remove large particles and bacteriovores. Collect the water filtrate in a carboy. </li>
 <li>Transfer 36 ml filtrate each into 3 sterile Eppendorf tubes (50 ml) containing 4 ml freshly prepared paraformaldehyde solution (PFA; 10%). Incubate for 2 hrs at room temperature to preserve cells. Collect cells onto 0.22-&mu;m-pore-size white membrane filters by vacuum filtration. Wash the filter by passing 10 ml phosphate-buffered saline (PBS) through the filter by vacuum filtration. Label the filters as negative controls and store them at -20&deg;C.</li> 
</ol>
Steps 1.3-1.4 are optional for establishing DOC-limited conditions.
<ol start="3">
 <li>Add a mixture of inorganic nitrogen and phosphorus (5 &mu;M NH4Cl, 5 &mu;M NaNO3, and 1&mu;M NaH2PO4 final concentration) into the carboy.</li> 
 <li>Incubate in the dark at in situ temperature for 48 hours with occasional agitations.</li>
</ol>
2. Microcosm establishment and incubation
<ol>
<li>Fill each of six 1-L glass flasks with 800 ml water sample from the carboy of step 1.4 to establish microcosms. Add BrdU (10 &mu;M, final concentration) to each microcosm. Mix well.</li>
<li>Add 1 ml model DOC compound solution into three of the microcosms, these will serve as DOC amendments. Add 1 ml sterile PBS into the three remaining microcosms, these will serve as no-addition controls. </li>
<li>Incubate all microcosms in an incubator shaker and incubate in the dark at in situ temperature while shaking at 100 rpm.</li>
<li>Collect 36 ml of water sample from each microcosm and transfer into 50 ml sterile Eppendorf tubes at time points of 0, 8, 16, and 24 hours. Immediately add 4 ml of fresh PFA (10%) to collection tubes and incubate for 2 hrs at room temperature to preserve the cells.</li>
<li>Filter preserved cells through 0.22-&mu;m-pore-size polycarbonate membrane filters. Wash the filters with 10 ml PBS. Proceed immediately to the next step or store the filters at -20&deg;C.</li>
</ol>
3. In situ immunodetection for BrdU Incorporation
<ol>
<li>Thaw the filters (DOC amended samples, no-addition controls and negative controls) at room temperature.</li>
<li>Apply 1 ml of lysozyme solution [10 mg/ml lysozyme egg white in 100 mM Tris, 50 nM EDTA (pH = 8)] 4 to cover bacterial cells on the filter. Incubate at room temperature for 30 minutes. Wash the filter by passing 10 ml PBS through it under suction. </li>
<li>Add 1 ml of proteinase K solution [2 mg/ml proteinase K in 100 mM Tris, 50 nM EDTA (pH = 8)] 4 to cover bacterial cells on the filter. Incubate at room temperature for 30 minutes. Wash the filter by passing 10 ml PBS through it under suction.</li>
<li>Add 1 ml exonuclease solution [exonuclease III (50 U/ml) in 5 mM MgCl2 and 50 mM Tris-HCl] 5 to cover bacterial cells on the filter. Incubate at 37 &deg;C for 30 minutes. Wash the filter by passing 10 ml PBS through it under suction.</li>
<li>Assemble frame-seal incubation chambers (Bio-Rad) according to the manufacturer's instructions. </li>
<li>Slice the filter into eighths using a sterile blade on an alcohol-cleaned dry surface.</li>
<li>Using forceps, place an eighth section of a filter into one assembled frame-seal incubation chamber (eight frame-seal chambers are needed for each filter sample). The side of the filter section that contains cells should face upwards. The moisture at the back of the filter will allow the filter stick to the slide. If filter becomes dry, apply a tiny drop (2 &mu;l) of diH2O in the center of the chamber before placing the filter section on the slide. </li>
</ol>
Steps 3.8-3.20 use the reagents from the ROCHE In Situ Cell Proliferation Kit, FLUOS following procedures modified from the manufacturer's instructions. Except for PBS, all reagents are provided within the kit.
<ol start="8">
<li>Apply enough incubation buffer (0.5% bovine serum albumen, 0.1% Tween20 in PBS,) to evenly cover the entire filter section in the incubation chamber without causing overflow once the seal is applied.</li> 
<li>Place a polyester frame cover over the incubation chamber frame. Press down to tightly seal the incubation chamber. Avoid air bubbles above the filter. Incubate the sealed chambers for 10 min at room temperature in the dark.</li>
<li>Remove the polyester seal and open the incubation chambers. (Note: opening the chambers sometimes will pull up the frame seal from the slide. In that case, prepare a new chamber for the following steps.)</li>
<li>Pipet out the incubation buffer from one corner of the chamber. Avoid contact with the filter.</li>
<li>Wash the filter by gently pipetting 100 &mu;l PBS in and out from the chamber 3 times.</li>
<li>Prepare anti-BrdU-FLUOS working solution following the steps recommended by the manufacturer immediately before use. </li>
<li>Pipet 120 &mu;l anti-BrdU-FLUOS working solution onto the filter, taking care to evenly cover the entire surface. </li>
<li>Reseal the chamber with a new polyester cover. Avoid air bubbles on top of the filter. Incubate the chamber in the dark at 37 &deg;C for 3 hours. This step will label BrdU incorporated DNA in situ with fluorescein isothiocyanate (FITC).</li>
<li>Remove the polyester cover and open the incubation chamber. Pipet out the anti-BrdU-FLUOS working solution. Wash the filter 3 times with PBS.</li>
<li>Transfer the filter from the incubation chamber to a sterile surface. Slice the filter section into small pieces using a sterile blade.</li>
<li>Transfer the filter pieces into 2 ml microcentrifuge tubes, each containing 1 ml PBS. Tightly cap the tube and seal with parafilm. Incubate at 37 &deg;C and 200 rpm for 10 min.</li> 
<li>Secure the tubes onto a vortexer and vortex at the maximum speed for 5 minutes. Pipet the supernatant into a sterile 15 ml Eppendorf tube. Repeat incubation and vortex steps for 5 more times. Combine the supernatant in the same 15 ml Eppendorf tube for each sample. Typically, 80% cells can be recovered in the suspension.</li>
<li>Store the supernatant with resuspended cells at 4 &deg;C. Sort within 2 days.</li>
</ol>

4. FACS analysis
A procedure for a BD FACSAria flow cytometer and corresponding software is described here. 
<ol>
<li>Optimize the setting of the flow cytometer following steps recommended by the manufacturer. This entails: adjusting the amplification control to set the required breakoff parameters and the sweet spot; optimizing the laser delay and area scaling factors for the experiment sheath pressure and optimizing settings for FSC and SSC voltages, FSC threshold, FSC fluorescence scaling, fluorescence PMT voltages, etc.</li>
<li>Run negative control samples on the flow cytometer (FCM) based on fluorescence intensity of FITC and side scatter (SSC). Increase the FITC threshold until no cells in the negative controls can be visualized by the FITC-SSC acquisition display.</li>
<li>Run no-addition control samples and examine the distribution pattern of 10,000 cells based on FITC and SSC acquisition. Define a gate to enclose all the cells and designate them as "low intensity cells" (LIs).</li>
<li>Run DOC-amended samples and examine the distribution pattern of 10,000 cells based on FITC and SSC. Some cells will appear in the preset LI gate. Define another gate to enclose the cells that have higher fluorescence intensity (HIs) than LIs. Acquire statistics to view the relative abundance of gated cells.</li>
<li>Sort HI cells into collection tubes containing 500 &mu;l PBS at "purify 1 drop" mode. Terminate sorting when the number of HIs reaches 500,000 counts.</li>
</ol>
5. Filter PCR amplification of 16S rRNA genes
Filter PCR procedures are modified from Kirchman et al. 6
<ol>
<li>Filter sorted cells onto a white, 0.22 &mu;m-pore-size, 25 mm-diameter, polycarbonate membrane filter. Trim off the edge of the filter that contains no bacterial cells using a sterile blade. Slice the filter into 8 equal sized pieces.</li> 
<li>Place a single filter piece into a PCR reaction tube, with the cells facing inward of the tube. Add 45 &mu;l PCR grade water into the PCR reaction tube. Submerge the filter entirely in the water. </li>
<li>Add 2 ROCHE illustra PuReTaq Read-To-Go PCR Beads into the PCR reaction tube, briefly vortex. Add 2 &mu;l each of the forward and reverse 16S rRNA gene primers (0.4 &mu;M final concentration for each primer), such as 27F and 1492R 7, into each of the PCR reaction tubes.</li> 
 <li>(Optional) Add 1&mu;l bovine serum albumin solution (BSA, final concentration is 30&mu;g/100 &mu;l) to the PCR reaction mixture to help adsorb amplification inhibitors. If one chooses not to add BSA, add 1&mu;l of water instead. </li>
 <li>Perform PCR amplification on a thermal cycler. A touch down PCR program is recommended, which has the annealing temperature sequentially decreasing from 62 to 52&deg;C by 1&deg;C per cycle for 11 cycles, followed by 15 cycles with annealing temperature of 52&deg;C. All cycles include denaturing (at 95&deg;C), annealing (at 62 to 52&deg;C), and extension (at 72&deg;C) steps of 50s duration. An initial 3-min denaturation and final 10-min extension step is also included in the PCR program.</li> 
 <li>Confirm PCR amplification by electrophoresis on an ethidium bromide-stained 1% agarose gel. Excise the PCR amplicons from the gel and clean with the QIAGEN QIAquick gel extraction kit.</li>
 <li>Perform two additional PCR amplifications for each sample, each time use a new filter section. After PCR gel purification, pool the amplicons of the same sample together. Purified 16S rDNA amplicons are now ready for a number of molecular analyses that allow taxonomic identification, such as clone library construction and sequencing.</li>
</ol>
6. Representative results: 
Representative results are described using a study of putrescine-degrading bacteria as an example. Water samples were collected from a coastal site of Georgia and processed following the procedures described above. FACS analysis revealed that putrescine addition induced development of a group of bacteria with high FITC fluorescence intensity, indicating high BrdU incorporation rate (Figure 1). These cells were designated as high-BrdU-incorporation cells (HIs) and were expected to contain mostly putrescine-degrading bacteria. HIs were missing in the no-addition controls, which only contained cells with lower levels of BrdU incorporation (LIs). LIs were expected to mainly contain bacterioplankton that were unable to use added putrescine. HIs were sorted into separated tubes and then collected onto membrane filters. 16S rRNA gene amplicons were obtained for high HI cells using filter PCR.
<img src="/files/ftp_upload/2855/2855fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow cytometric analysis of no-addition control and model-compound-amended (putrescine as an example here) samples collected after 24 h of incubation. Cell distribution analysis was based on (1) fluorescence intensity of FITC labeling (x-axis), which is positively related to level of BrdU incorporation, and (2) side scatter (SSC, y-axis), which is positively related to cell size. Gate notation is based on level of BrdU incorporation, (HI, high-BrdU-incorporation; LI, low-BrdU-incorporation). The relative percentage of HI and LI cells are shown in corresponding gates.

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, 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=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, 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=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <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+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

No conflicts of interest declared.

Our approach couples BrdU incorporation, FACS and 16S rDNA analysis to allow species-level identification of bacterioplankton that metabolize individual DOC components in aquatic environments. The BrdU incorporation assay labels bacterial cells based on metabolic activities, which allows analysis only on active bacteria and thus does not include dormant cells. In our approach, BrdU incorporation is in situ immunodetected and bacteria that have different levels of BrdU incorporation are subsequently visualized, g...

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>BrdU</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>B5002-5G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lysozyme</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>L6876-5G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>P2308-25MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>In Situ Cell Proliferation Kit, FLUOS</td>
 <td>Kit</td>
 <td>Roche</td>
 <td>11810740001</td>
 <td>Consume more of Anti-BrdU-FLUOS and Incubation buffer per reaction than suggested by the manufacturer.</td>
 </tr>
 <tr>
 <td>Frame-Seal Incubation Chambers</td>
 <td>Material</td>
 <td>Bio-Rad</td>
 <td>SLF-1201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polycarbonate Membrane Filters (142-mm-diameter, 1.0 &mu;m-pore-size)</td>
 <td>Material</td>
 <td>Millipore</td>
 <td>FALP14250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polycarbonate Membrane Filters (25-mm-diameter, 0.2 &mu;m-pore-size)</td>
 <td>Material</td>
 <td>Millipore</td>
 <td>FGLP02500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>illustra PuReTaq Ready-To-Go PCR Beads</td>
 <td>Kit</td>
 <td>GE Healthcare</td>
 <td>27-9559-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>QIAquick gel extraction kit</td>
 <td>Kit</td>
 <td>QIAGEN</td>
 <td>28704</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FailSafe PCR System</td>
 <td>Kit</td>
 <td>EPICENTRE</td>
 <td>FS99060</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

bromodeoxyuridine-brdu-labeling-subsequent-fluorescence-activated

Research

JoVE Journal

Biology

20.9K vues.  Kent State University. Bactérioplancton environnement sont incubés avec un modèle de carbone organique dissous (DOC) composé et un réactif de marquage de l'ADN, bromodésoxyuridine (BrdU). Ensuite, DOC-dégradants cellules sont séparées de la communauté en vrac en fonction de leur incorporation de BrdU élevée en utilisant la fluorescence tri cellulaire (FACS). Ces cellules sont ensuite identifiés par des analyses moléculaires ultérieures.

Vidéo: Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

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 ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...