This work was funded by the Smith Family Award for Excellence in Biomedical Research and NIH RO1 AI083426-01 (F.W.). Patrick Maschmeyer and Melanie Flach are supported by PhD fellowships from the Boehringer Ingelheim Foundation.

C57BL/6 mice should be used at ~20 weeks of age or older. The use of male mice weighing 25-30g is recommended. The yield of stellate cells can be increased by feeding mice a vitamin A-enriched diet for 2 months prior to stellate cell isolation. Approximately 2x105 hepatic stellate cells can be purified from the liver of one C57BL/6 mouse, whereas the yield of stellate cells from Balb/c mice is considerably higher. The following protocol is adjusted to 5 mice. Animal care and experimentation were performed in accordance with approved Institutional Animal Care and Use Committee protocols. 


1. In situ perfusion of mouse livers with digestive enzymes 
<ol>
 <li>Warm the SC1 buffer and enzyme perfusion solutions in a 37&deg;C water bath.</li>
 <li>Mount a winged infusion set onto the silicone tube of a peristaltic pump.</li>
 <li>Calibrate the pump with PBS to obtain a laminar flow of 6.5ml/min, which is the flow rate that will be used for the in situ perfusion of the liver. Equilibrate the silicone tube with SC1 solution.</li>
 <li>Anesthetize the mouse by intraperitoneal injection of Ketamine (90 mg/kg) and Xylazine (10 mg/kg) solution and test for loss of reflexes to ensure deep narcotization.</li>
 <li>Fix the mouse in supine position on a suitable base. </li>
 <li>Use scissors and forceps to perform a longitudinal incision in the abdominal skin and expose the peritoneum.</li>
 <li>Carefully open the peritoneum and move the intestines out of the abdominal cavity to the left side of the animal in order to expose the portal vein.</li>
 <li>Insert the cannula of the infusion set into the portal vein. For this step, the use of a stereomicroscope is recommended.</li>
 <li>Start the perfusion of the liver with 30ml SC1 solution. Open the vena cava inferior immediately after initiating the flow. Successful flushing of the liver is indicated by a loss of color of the liver tissue.</li>
 <li>Perfuse the liver with 30ml Pronase E solution. Upon successful digestion, the liver lobes will swell, and the lobules will appear distinct through the capsule.</li>
 <li>Perfuse the liver with 30ml Collagenase P solution. The liver at this point has lost its shape and looks atonic and amorphous.</li>
 <li>Carefully separate the liver from the diaphragm and surrounding organs and store it in 70ml SC2 solution on ice.</li>
 <li>Repeat the procedure for additional mice.</li>
</ol>





2. In vitro digestion of mouse livers 

All further working steps should be carried out under sterile conditions in a laminar flow hood.

<ol>
 <li>Cut the livers into pieces of approximately 2x2x2mm3 using sharp scissors. </li>
 <li>Combine the suspension of 70ml SC2 including the liver pieces with 50ml of Pronase E-Collagenase P solution and add 1ml of DNase I solution.</li>
 <li>Digest the livers for 20min at 37&deg;C while stirring.</li>
</ol>
3. Density gradient centrifugation 
<ol>
 <li>Filter the cell suspension through 70&mu;m cell strainers into 6 50ml Falcon tubes and add up to 50ml with SC2 buffer. Centrifuge for 10min at 600g and 4&deg;C.</li>
 <li>Carefully aspirate 40ml of the supernatant, then add 150&mu;l DNase I solution to each tube and resuspend the cells. </li>
 <li>Pool the cell suspensions into 4 50ml Falcon tubes and wash with GBSS-B, centrifuge for 10min at 600g and 4&deg;C.</li>
 <li>Carefully aspirate as much of the supernatant as possible without disturbing the pellet and add 150&mu;l DNase I solution to each tube. Resuspend the cell pellets in 10ml GBSS-B per tube. </li>
 <li>Pool the cells into 2 50ml Falcon tubes and add GBSS-B to a total volume of 36ml per tube. Add 14ml Nycodenz solution to each tube and mix well.</li>
 <li>Transfer 10ml of the cell suspension into one 12ml gradient centrifugation tube, resulting in 10 tubes in total. Gently overlay the cell suspension with 1.5ml GBSS-B per tube. </li>
 <li>Centrifuge the gradients for 15min at 1500g and 4&deg;C without brake. Subsequently, hepatocytes will be pelleted at the bottom of the tube whereas stellate cells are found in the interphase as a white ring.</li>
 <li>Carefully harvest the interphase containing the stellate cells and wash them with GBSS-B; centrifuge for 10min at 600g and 4&deg;C.</li>
 <li>Aspirate the supernatant and resuspend the cells in 20ml DMEM supplemented with 10% heat-inactivated FBS, 1% Penicillin/Streptomycin, 2mM L-Glutamine, 1mM sodium pyruvate, and 10mM HEPES. Transfer the cells into tissue culture flasks with a concentration of 2x104 cells/cm2. Incubate the cells at 37&deg;C and 5% CO2. </li>
 <li>Change the media as soon as the hepatic stellate cells are adherent (approximately 2h after the preparation) to wash off dead cells and cell debris. </li>
 <li>On the following day, the stellate cells should develop their characteristic star-shaped morphology with perinuclear vitamin A-storing lipid vesicles. </li>
 <li>For subsequent experiments, hepatic stellate cells can easily be detached from non-coated plastic surfaces using mild enzymatic solutions such as Accutase, e.g.</li>
</ol>


4. Representative Results:
Following the preparation of hepatic stellate cells using the protocol provided, the purity of the isolated population can be tested considering three major characteristics of this cell type, such as star-like shape, perinuclear lipid droplets, and expression of glial fibrillary acidic protein (GFAP). Representative pictures for the characteristic appearance of hepatic stellate cells 2h after cell isolation, as well as on day 1 and 3 of in vitro culture are depicted in Figure 1. Figure 2 shows a representative immunofluorescence staining for GFAP in hepatic stellate cells, which have been cultured for 3 days following cell isolation. Hepatic stellate cells differentiate into myofibroblastic cells during in vitro culture. A characteristic hallmark of those activated stellate cells is the expression of alpha smooth muscle actin (&alpha;SMA). Figure 3 shows immunofluorescence staining for aSMA and myosin IIA in activated stellate cells on day 7 of in vitro culture. 



<img src="/files/ftp_upload/2710/2710fig1.jpg" alt="Figure 1" /> 
Figure 1. Characteristic morphology of hepatic stellate cells. Stellate cells were isolated from mouse livers using the protocol provided. The images depict stellate cells 2h after cell isolation (a, b), as well as on day 1 (c) and day 3 (d) of in vitro culture. Hepatic stellate cells exhibit high amounts of lipid vesicles at perinuclear sites and acquire their distinctive astral-like morphology during the first days of in vitro culture (Magnification 200x).



<img src="/files/ftp_upload/2710/2710fig2.jpg" alt="Figure 2" /> 
Figure 2. Hepatic stellate cells specifically express GFAP in the liver. Stellate cells were isolated and immunofluorescently stained for GFAP (red) on day 3 of in vitro culture. Cell nuclei are depicted in blue (Hoechst stain).


<img src="/files/ftp_upload/2710/2710fig3.jpg" alt="Figure 3" /> 
Figure 3. Liver stellate cells differentiate into myofibroblasts. Hepatic stellate cells isolated from C57BL/6 mice were cultured for 7 days. Subsequently, they were transferred to chamber slides and stained for aSMA (shown in red) and myosin IIA (depicted in green). The cell nuclei were counterstained with Hoechst (blue). 
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">SC1 Buffer</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>190mg</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>900mg</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>10 ml of 1M stock solution</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>400mg</td>
 </tr>
 <tr>
 <td>Na2HPO4 x 2 H2O</td>
 <td>151mg</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>8g</td>
 </tr>
 <tr>
 <td>NaH2PO4 x H2O</td>
 <td>78mg</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>350mg</td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>6mg</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>fill up to 1l</td>
 </tr>
 <tr>
 <td colspan="2">SC2 Buffer</td>
 </tr>
 <tr>
 <td>CaCl2 x 2H2O</td>
 <td>560mg</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>10ml of 1M stock solution</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>400mg</td>
 </tr>
 <tr>
 <td>Na2HPO4 x 2 H2O</td>
 <td>151mg</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>8g</td>
 </tr>
 <tr>
 <td>NaH2PO4 x H2O</td>
 <td>78mg</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>350mg</td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>6mg</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>fill up to 1l</td>
 </tr>
 <tr>
 <td colspan="2">GBSS-A Buffer</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>370mg</td>
 </tr>
 <tr>
 <td>CaCl2 x 2H2O</td>
 <td>225mg</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>991mg</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>30mg</td>
 </tr>
 <tr>
 <td>MgCl2 x 6 H2O</td>
 <td>210mg</td>
 </tr>
 <tr>
 <td>MgSO4 x 7 H2O</td>
 <td>70mg</td>
 </tr>
 <tr>
 <td>Na2HPO4 x 2 H2O</td>
 <td>75mg</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>227mg</td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>6mg</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>fill up to 1l</td>
 </tr>
 <tr>
 <td colspan="2">GBSS-B Buffer</td>
 </tr>
 <tr>
 <td>CaCl2 x 2H2O</td>
 <td>225mg</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>991mg</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>370mg</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>30mg</td>
 </tr>
 <tr>
 <td>MgCl2 x 6 H2O</td>
 <td>210mg</td>
 </tr>
 <tr>
 <td>MgSO4 x 7 H2O</td>
 <td>70mg</td>
 </tr>
 <tr>
 <td>Na2HPO4 x 2 H2O</td>
 <td>75mg</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>8g</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>227mg</td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>6mg</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>fill up to 1l</td>
 </tr>
 <tr>
 <td colspan="2">Pronase E Perfusion Solution</td>
 </tr>
 <tr>
 <td>Pronase E</td>
 <td>100mg (4000 PU/mg min) </td>
 </tr>
 <tr>
 <td>SC2 Buffer</td>
 <td>200ml</td>
 </tr>
 <tr>
 <td colspan="2">Collagenase P Perfusion Solution</td>
 </tr>
 <tr>
 <td>Collagenase P</td>
 <td>85mg (1.78 U/mg lyo)</td>
 </tr>
 <tr>
 <td>SC2 Buffer</td>
 <td>200ml</td>
 </tr>
 <tr>
 <td colspan="2">Pronase E-Collagenase P Solution</td>
 </tr>
 <tr>
 <td>Pronase E</td>
 <td>50mg (4000 PU/mg min) </td>
 </tr>
 <tr>
 <td>Collagenase P</td>
 <td>85mg (1.78 U/mg lyo)</td>
 </tr>
 <tr>
 <td>SC2 Buffer</td>
 <td>50ml</td>
 </tr>
 <tr>
 <td colspan="2">DNase I Solution</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>6mg (ca. 2000U/mg)</td>
 </tr>
 <tr>
 <td>GBSS-B</td>
 <td>3ml</td>
 </tr>
 <tr>
 <td colspan="2">Nycodenz Solution</td>
 </tr>
 <tr>
 <td>Nycodenz</td>
 <td>8g</td>
 </tr>
 <tr>
 <td>GBSS-A</td>
 <td>28ml</td>
 </tr>
 </tbody>
</table>
Table 1. Buffers and enzyme solutions required for isolation of hepatic stellate cells. The pH of all buffers should be adjusted to 7.3-7.4. Furthermore, sterile filtration of all buffers is recommended. It is important to adapt the amount of enzyme used according to the given enzymatic activity.

<ol>
	<li>Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

No conflicts of interest declared.

Hepatic stellate cells regulate essential physiological and pathophysiological processes in the liver. Moreover, stellate cells possess antigen presenting properties, rendering them an important component of the hepatic immune response. Although hepatic stellate cells comprise 10-15% of the total cell number in the liver, isolation of these cells is challenging due to their localization in the perisinusoidal space of Disse.

 Here we present a straightforward method to isolate hepatic ...

<table border="1">
<tbody>
 <tr>
 <th>Name Of Reagent</th>
 <th>Company</th>
 <th>Catalog No.</th>
 </tr>
 <tr>
 <td>70&mu;m Cell Strainer</td>
 <td>BD</td>
 <td>352350</td>
 </tr>
 <tr>
 <td>CaCL2 x 2H2O</td>
 <td>Sigma-Aldrich</td>
 <td>C3306</td>
 </tr>
 <tr>
 <td>Collagenase P</td>
 <td>Roche</td>
 <td>11249002001</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11960</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td>10104159001</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Cellgro</td>
 <td>21-031-cv</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Fluka</td>
 <td>3777</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Gibco</td>
 <td>16000</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G7528</td>
 </tr>
 <tr>
 <td>Gradient Centrifugation Tubes</td>
 <td>Greiner Bio-One</td>
 <td>163160</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Gibco</td>
 <td>15630</td>
 </tr>
 <tr>
 <td>KCL</td>
 <td>Sigma-Aldrich</td>
 <td>P9541</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>P9791</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 </tr>
 <tr>
 <td>MgCl2 x 6H2O</td>
 <td>Fluka</td>
 <td>63068</td>
 </tr>
 <tr>
 <td>MgSO4 x 7H2O</td>
 <td>Sigma-Aldrich</td>
 <td>M2773</td>
 </tr>
 <tr>
 <td>Na2HPO4 x 2H2O</td>
 <td>Fluka</td>
 <td>71643</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 </tr>
 <tr>
 <td>NaH2PO4 x H2O</td>
 <td>Fluka</td>
 <td>71507</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Fluka</td>
 <td>71628</td>
 </tr>
 <tr>
 <td>Nycodenz</td>
 <td>Accudenz AG</td>
 <td>AN7050/BLK</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Gibco</td>
 <td>15140</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Cole Parmer</td>
 <td>HV-7523-70</td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>Sigma-Aldrich</td>
 <td>P4633</td>
 </tr>
 <tr>
 <td>Pronase E</td>
 <td>Calbiochem</td>
 <td>7433-2</td>
 </tr>
 <tr>
 <td>Silicone Tube</td>
 <td>Masterflex</td>
 <td>HV-96440-14</td>
 </tr>
 <tr>
 <td>Sodium Pyruvate</td>
 <td>Gibco</td>
 <td>11360</td>
 </tr>
 <tr>
 <td>Tissue culture flask (25cm2)</td>
 <td>BD</td>
 <td>353108</td>
 </tr>
 <tr>
 <td>Winged Infusion Set</td>
 <td>Terumo</td>
 <td>1SV27EL</td>
 </tr>
 </tbody>
</table>

seven steps to stellate cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Research

JoVE Journal

Immunology and Infection

33.5K Views.  Harvard Medical School. Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Video: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...