Danilo Maddalo is a recipient of a research fellowship as Young Investigator Group (YIG) from the Karlsruhe Institute of Technology.

1. Seeding the cells

<ol> 
 <li>Before starting, warm up the culture medium, the PBS 1X and the trypsin in a water bath at 37&#730;C</li>
 <li>Take the cells out of the incubator and aspirate the medium.</li>
 <li>Gently apply 5 mL of PBS 1X on the cells to wash them.</li>
 <li>Aspirate the PBS 1X and apply 1 mL of trypsin containing 0,025% EDTA.</li>
 <li>Gently rotate the plate to have an even distribution of the trypsin.</li>
 <li>Place the cells in the incubator at 37&#730;C for 5-10 minutes (depending on the cell type). From time to time check if cells are detached by shaking the plate.</li>
 <li>Resuspend the cells in 10-20 mL of medium and collect them in a 50 mL falcon tube.</li>
 <li>Count the cells using a Beckman counter. The number of cells should be enough to ensure a confluence of approximately 60-70%.</li>
 <li>Plate the cells and allow them to seed for 6-8 hours.</li>
 
</ol>

2. Transfection

<ol>
 <li>Prior to transfection, warm up an aliquot of serum-free medium in a water bath at 37&#730;C.</li>
 <li>Bring the X-treme Gene 9 to room temperature.</li>
 <li>Pipet the serum free medium in 2 eppendorf tubes (1 tube= 1 transfection).</li>
 <li>Pipet the X-treme Gene 9 in the serum free medium paying attention that the tip does not touch the plastic wall of the eppendorf and incubate for 5 minutes. In the meantime prepare two DNA mixes: in one tube mix together the plasmids for renilla, firefly and an empty vector control, in another tube renilla, firefly and the molecular chaperone. </li>
 <li>Add the DNA mix to each eppendorf containing serum free medium + X-treme Gene 9, vortex and incubate at room temperature for 20 minutes.</li>
 <li>Add dropwise each mix onto each plate and incubate for 24 hours at 37&deg;C and 5% CO2</li>
</ol>

3. Splitting the cells

<ol>
 <li>24 hours after transfection, trypsinize the cells as described in 1.4 &ndash; 1.6 and dilute them to a final confluency of 60-70% in a 6 -well plate.</li>
 <li>Prepare one plate for the non-shocked control and as many plates as the recovery time points that have to be analyzed, as showed in Fig. 2.</li>
 
</ol>

4. Heat shock

<ol>
 <li>For heat shock, pre-warm serum containing medium in a water bath at 37&#730;C.</li>
 <li>Prepare &lsquo;heat shock buffer&rsquo; by diluting in serum containing medium cycloheximide to a final concentration of 20 &micro;g/ml and MOPS to 20 mM.</li>
 <li>Now take out the plates from the incubator and aspirate the medium.</li>
 <li>Add to each well 1 mL of &lsquo;Heat shock buffer&rsquo;, and incubate for 30 minutes at 37&#730;C and 5% of CO2 (include also the reference plate in this treatment). This will stop de novo protein synthesis.</li>
 <li>In the meantime switch on a water bath and set the temperature at 45&#730;C.</li>
 <li>After incubation, seal the lid of the plates with Parafilm previously cut in stripes. The reference plate (100% of refolfed firefly) is left in the incubator.</li>
 <li>Incubate the plates at 45&#730;C for 30 minutes.</li>
 <li>Remove the plates from the water bath, remove the parafilm and put the plates back to the incubator to allow recovery.</li>
 <li>At each time point harvest the cells and wash them in PBS 1X by centrifuging at 2000Xg for 1 minute at 4&#730;C.</li>
</ol>

5. Cell lysis and luciferase assay

<ol>
 <li>For an efficient cell lysis, snap freeze the cell pellet in liquid nitrogen.</li>
 <li>Add 200 &micro;l of passive lysis buffer diluted 1:5 in double distilled water in a glass tube.</li>
 <li>Add 30 &micro;l of the cell lysate in a 96 well plate. Each sample is pipetted in triplicate.</li>
 <li>Only the &lsquo;non shocked&rsquo; reference samples will be used to read the renilla luciferase activity, since this step is used to check equal transfection efficiency.</li>
 <li>Prepare the solutions for the assay. For the luciferin solution dilute luciferin to a final concentration of 0.2 mM in Gly-Gly buffer. For the reaction buffer dilute DTT and ATP to 1mM final concentration in Gly-Gly buffer. For coelenterazine reaction buffer, dilute coelenterazine to a final concentration of 0.2 &micro;M in coelenterazine buffer. Keep luciferin solution and coelenterazine reaction buffer light protected.</li>
 <li>Now place the 96-well plate in the luminometer and start the program &lsquo;Wallac 1420 Workstation&rsquo;.</li>
 <li>Introduce the Pump1 into the falcon tube containing the coelenterazine solution. Close the lid so no light can come in contact with the tube.</li>
 <li>Choose from the menu the option &lsquo;Dispenser maintenance&rsquo; and tick Pump1.</li>
 <li>Select the option &lsquo;Fill&rsquo;.</li>
 <li>To edit the plate layout choose the option &lsquo;Explore protocol and results&rsquo; on the menu. Select the protocol and double click on it. Now select the wells to be read.</li>
 <li>Go back to &lsquo;Wallac Manager&rsquo; and click &lsquo;Start&rsquo;. The reaction between the renilla luciferase and its substrate coelenterazine emits light with a peak wavelength of&nbsp; 482 nm.</li>
 <li>After the reading, go on &lsquo;Dispenser maintenance&rsquo; and select &lsquo;Empty&rsquo; and Pump1 to release all the residual solution back to the tube.</li>
 <li>Now move the tube to a falcon tube containing water and tick &lsquo;Flush&rsquo;.</li>
 <li>After the flushing step, empty the tube by selecting &lsquo;Empty&rsquo;.</li>
 <li>Place the tube from Pump1 in the Reaction buffer falcon tube and the one from Pump2 in the luciferin falcon tube .</li>
 <li>Change the plate layout.</li>
 <li>Select &lsquo;Fill&rsquo; for Pump1 and Pump2 from the &lsquo;Dispenser Maintenance&rsquo; menu.</li>
 <li>Select the protocol and start the reading. The reaction between the Firefly luciferase and its substrate luciferin emits light with a peak wavelength of 560 nm.</li>
 <li>At the end of the reading repeat the procedure Empty-Flush-Empty for Pump1 and Pump2.</li>
 <li>Results are now available in the &lsquo;Wallac 1420 Explorer&rsquo;.</li>
 <li>Each experiment is associated to an assay number, username, begin and end date.</li>
 <li>Results can now be exported as an Excel file.</li>
 <li>For calculations, cells are divided into three groups: 1. non-shocked reference samples (set as 100% of refolded firefly luciferase); 2. cells without molecular chaperone and heat shocked; 3. cells with molecular chaperone and heat shocked.</li>
</ol>


6. Representative Results

Protein refolding is directly related to the time of recovery therefore to test if the system is working properly, an assay has to be performed collecting the cells at different time points. A direct correlation time/percentage of refolding indicates that the experiment has been properly performed and therefore represents a limiting factor. However it should be always considered that refolding after heat shock is a saturating event and therefore a proper time course analysis should be performed before starting any experiment.








<img src="/files/ftp_upload/3540/3540fig1.jpg" alt="Figure 1"/> Figure 1.&nbsp;

Overview of the experiment. In vivo refolding assay requires 3 to 4 days to be completed. On day 1 cells are seeded and transfected. The following day cells are splitted into a smaller format and on day 3 they are heat shocked. At this point is possible to perform the luciferase assay right after harvesting the cells or to save the cell pellet at -80&deg;C and perform the assay in another moment 

<img src="/files/ftp_upload/3540/3540fig2.jpg" alt="Figure 2"/> Figure 2.&nbsp;
Cell splitting. On day 2 cells are split into a 6-well plate. Each transfection will be diluted in a well in order to have all the transfection on the same plate. Each plate will correspond to a particular recovery time plus a non-shocked reference plate.

<img src="/files/ftp_upload/3540/3540fig3.jpg" alt="Figure 3"/> Figure 3.&nbsp;
Representative good result. A. Percentage of refolding directly measured by firefly luciferase activity. Each bar chart represents the percentage of refolding in absence (black bars) and in presence (red bars) of a molecular chaperone at different recovery time points. B. Correlation between time and refolding in absence (black line) and in presence (red line) of chaperone. The r squared values indicate good correlation.

In Fig.3A is shown a representative results where cells were collected 15, 30, 45, 60 and 120 minutes after heat shock in absence (black bars) and in presence (red bars) of molecular chaperone. Percentage of refolded luciferase increases with prolonged recovery time and with the transfection of the molecular chaperone. The correlation between refolding and time for the control (Fig. 3B, black line) and the molecular chaperone-transfected cells (Fig. 3B, red line) is shown.

<img src="/files/ftp_upload/3540/3540fig4.jpg" alt="Figure 4"/> Figure 4.&nbsp;
Representative bad result. A. Percentage of refolding directly measured by luciferase activity. Each bar chart represents the percentage of refolding in absence (black bars) and in presence (red bars) of a molecular chaperone at different recovery time points. B. Correlation between time and refolding in absence and in presence (red line) of chaperone. The r squared values indicate bad correlation.

Figure 4A. shows a representative result of an experiment not properly performed. Both the control (black bars) and the molecular chaperone-transfected cells (red bars) did not show an increase in protein refolding with the time. In addition, transfection of the molecular chaperone did not result in an increase of refolding. This poor correlation is confirmed in Fig. 4B showing an r squared value of 0.6619 and 0.1882 respectively for the control (black line) and the molecular chaperone-transfected cells (red line).

<ol>
	<li>Bukau, B., Weissman, J., Horwich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperones+and+protein+quality+control.">Molecular chaperones and protein quality control.</a> Cell. 125, 443-443 (2006).</li><li>Ritossa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+the+heat+shock+response.">Discovery of the heat shock response.</a> Cell Stress Chaperones. 1, 97-97 (1996).</li><li>Hendrick, J. P., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+functions+of+heat-shock+proteins.">Molecular chaperone functions of heat-shock proteins.</a> Annu. Rev. Biochem. 62, 349-349 (1993).</li><li>Fuqua, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+proteins+and+drug+resistance.">Heat shock proteins and drug resistance.</a> Breast. Cancer. Res. Treat. 32, 67-67 (1994).</li><li>Guzhova, I., Margulis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperone+as+a+survival+factor+in+cell+pathology.">Hsp70 chaperone as a survival factor in cell pathology.</a> Int. Rev. Cytol. 254, 101-101 (2006).</li><li>Whitesell, L., Lindquist, 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=HSP90+and+the+chaperoning+of+cancer.">HSP90 and the chaperoning of cancer.</a> Nat. Rev. Cancer. 5, 761-761 (2005).</li><li>Konstantinopoulos, P. A., Papavassiliou, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17-AAG:+mechanisms+of+antitumour+activity.">17-AAG: mechanisms of antitumour activity.</a> Expert. Opin. Investig. Drugs. 14, 1471-1471 (2005).</li><li>Galluzzi, L., Giordanetto, F., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+HSP70+for+cancer+therapy.">Targeting HSP70 for cancer therapy.</a> Mol. Cell. 36, 176-176 (2009).</li><li>Ozacmak, V. H., Barut, F., Ozacmak, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+provides+neuroprotection+by+reducing+oxidative+stress+and+HSP70+expression+during+chronic+cerebral+hypoperfusion+in+ovariectomized+rats.">Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats.</a> J. Pineal. Res. 47, 156-156 (2009).</li><li>Schroder, H., Langer, T., Hartl, F. U., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK,+DnaJ+and+GrpE+form+a+cellular+chaperone+machinery+capable+of+repairing+heat-induced+protein+damage.">DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.</a> EMBO. J. 12, 4137-41 (1993).</li><li>Thulasiraman, V., Matts, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geldanamycin+on+the+kinetics+of+chaperone-mediated+renaturation+of+firefly+luciferase+in+rabbit+reticulocyte+lysate.">Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.</a> Biochemistry. 35, 13443-13443 (1996).</li><li>Howarth, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp40+molecules+that+target+to+the+ubiquitin-proteasome+system+decrease+inclusion+formation+in+models+of+polyglutamine+disease.">Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.</a> Mol. Ther. 15, 1110-1110 (2007).</li><li>Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bag1+functions+in+vivo+as+a+negative+regulator+of+Hsp70+chaperone+activity.">Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.</a> Mol. Cell. Biol. 20, 1083-1083 (2000).</li></ol>

The authors have nothing to disclose.

In this work a protocol to measure intracellular refolding activity of molecular chaperones is presented. The whole assay can be performed in 3 to 4 days as shown by the overview in Fig. 1.

The robustness and linearity of the light signal produced by the firefly and the renilla luciferase represent a solid base for the reproducibility of the protocol. 

The critical step of the assay is the choice of an efficient transfection reagent to ensure the ...

For this experiment a Luminometer form Perkin Elmer '1420 Luminescence Counter' Vector Light&prime; was used

Programs for the Luminometer:

Renilla: Pump 1, 100 &mu;l injection

Reaction Buffer: Pump1, 70 &mu;l injection

Luciferin: Pump2, 30&mu;l injection


For the buffers prepare these stock solutions in double distilled water:

<ul style="list-style:none">
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1M MgSO4 : 246,48g MgSO4*7H2O in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0,5M EGTA: 190,18g EGTA in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1M KH2PO4: 136,09g KH2PO4 in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1M K2HPO4: 228,23g K2HPO4*3H2O in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5M NaCl: 292,2g NaCl in 1 liter</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0,5M EDTA: 186,12g EDTA*2H2O in 1 liter&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;</li>
</ul>

GLY-GLY-buffer: 

<ul style="list-style:none">
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;25mM Glycylglycin </li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;15mM MgSO4</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4mM EGTA</li>
</ul>

For 1 liter use 3,3g of Glycylglycin, 15ml of a 1M MgSO4 solution and 8ml of a 0,5M EGTA solution. Solve in water, adjust the pH to 7,8 and bring to a total volume of 1 liter.


Reaction buffer for Renilla/Coelenterazine buffer: 

<ul style="list-style:none">
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;13,4mM KH2PO4</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;86,6mM KH2PO4</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0,5M NaCl</li>
 <li>- &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1mM EDTA</li>
</ul>

For 1 liter use 13,4ml of a 1M KH2PO4 solution, 86,6ml of a 1M K2HPO4 solution, 100ml of a 5M NaCl solution and 2ml of a 0,5M EDTA solution. 
<table border="1">
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalog/Product Number:</td>
 </tr>
 <tr>
 <td>DMEM (Dulbecco&rsquo;s Modified Eagle Medium 1X)</td>
 <td>GIBCO</td>
 <td>41966029</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum Gold</td>
 <td>PAA</td>
 <td>A15-151</td>
 </tr>
 <tr>
 <td>X-treme Gene 9 
 DNA Transfection Reagent</td>
 <td>Roche</td>
 <td>06365809001</td>
 </tr>
 <tr>
 <td>PBS 
 Dulbecco&rsquo;s Phosphate Buffered Saline 1X</td>
 <td>GIBCO</td>
 <td>14190-094</td>
 </tr>
 <tr>
 <td>MOPS 
 3-(N-Morpholino)Propanesulfonic acid</td>
 <td>SIGMA-ALDRICH</td>
 <td>M1254</td>
 </tr>
 <tr>
 <td>Cycloheximide</td>
 <td>SIGMA-ALDRICH</td>
 <td>C7698</td>
 </tr>
 <tr>
 <td>Passive Lysis Buffer 5X</td>
 <td>Promega</td>
 <td>E194A</td>
 </tr>
 <tr>
 <td>
 Glycylglycin 
 EGTA 
 MgSO4 (MgSO4*7H2O)</td>
 <td>SIGMA-ALDRICH 
 Roth 
 Roth</td>
 <td>G7278 
 3054.2 
 P027.1 </td>
 </tr>
 <tr>
 <td>KH2PO4 
 K2HPO4 (K2HPO4*3H2O) 
 NaCl 
 EDTA</td>
 <td>Roth 
 Roth 
 Roth 
 Roth</td>
 <td>3904.1 
 6878.2 
 3957.1 
 8043.1</td>
 </tr>
 <tr>
 <td>Luciferin Firefly</td>
 <td>Biosynth</td>
 <td>L8200</td>
 </tr>
 <tr>
 <td>Coelenterazine</td>
 <td>Biosynth</td>
 <td>C7000</td>
 </tr>
 <tr>
 <td>DTT (Dithiothreitol)</td>
 <td>Roth</td>
 <td>6908.2</td>
 </tr>
 <tr>
 <td>ATP (Adenosine Triphosphate)</td>
 <td>Roche</td>
 <td>10519987001</td>
 </tr>
</table>

intracellular refolding assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

Intracellular Refolding Assay

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Research

JoVE Journal

Biology

14.1K vues.  Karlsruhe Institute of Technology. Dans ce protocole, une méthode pour mesurer le repliement des protéines intracellulaires, après choc thermique est décrite. Cette méthode peut être utilisée pour étudier foldases comme chaperons moléculaires et leurs co-facteurs ou des composés capables d'influencer leur activité. Firefly activité luciférase est utilisée en tant que reporter pour mesurer l'activité de repliement chaperon.

Vidéo: Intracellular Refolding Assay

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...