This work was supported by the Uehara Memorial Foundation of Japan (to ES) as well as the Whitehall Foundation, the National Institute of Neurological Disorders and Stroke and a Stein-Oppenheimer Endowment Award (to BSK).

EXPERIMENTAL PROCEDURES
The experimental procedure consists of two key parts that are described in a step wise manner below.
Part 1: PREPARING HIPPOCAMPAL ASTROCYTE CULTURES
Briefly, mixed hippocampal astrocyte-neuron cultures were prepared using a well established protocol1,2,3. We optimized the procedure to yield healthy cultured astrocytes. All the procedures listed below should be carried out in a sterile environment such as a laminar flow hood.
Preparing coverslips
<ul>
	<li>22 mm coverslips (VWR, 48380-068)</li>
	<li>Poly-D-Lysine (PDL, Sigma P0899), aliquots (1 mg/ml)</li>
	<li>Laminin aliquots (20 &#956;g/ml): add 49 ml of sterile water to 1 mg/ml laminin solution (Sigma L2020), make 1.2 ml aliquots and store at -20&#176;C</li>
</ul>
<ol>
	<li>Dissolve PDL in sterile water (50 &#956;g/ml)</li>
	<li>Autoclave the coverslips, rinse with sterile water and place in PDL (20 ml for 100 coverslips). Leave at room temperature overnight.</li>
	<li>Replace PDL with sterile water (3 extensive washes). Then dry the coverslips and store at 4&#176;C.</li>
	<li>The day before plating the astrocytes, place individual coverslips in a sterile well on a 6-well plate (Multiwell Flat-Bottom Plates with Lids, Sterile from BD Bioscience). Put 400 &#956;l of laminin on each coverslip and incubate overnight, in the incubator to avoid evaporation.</li>
</ol>

Dissection
Dissection media:
<ul>
	<li>500 ml Earle&#8217;s Balanced Salt Solution (EBSS) (1X), liquid (Invitrogen, 14155-063)</li>
	<li>5 ml HEPES solution (Sigma, H0887)</li>
</ul>

Hippocampal media:
<ul>
	<li>412.5 ml Minimum Essential Medium (MEM) (1X), liquid Contains Earle&#39;s salts, but no L-glutamine or phenol red (Invitrogen, 51200-038)</li>
	<li>10 ml glucose (Sigma, D-(+)-GLUCOSE ANHYDROUS SIGMA ULTRA G7528) 1 M in MEM</li>
	<li>5 ml Penicillin-Streptomycin (Invitrogen, 15140-122)</li>
	<li>5 ml Sodium pyruvate solution (Sigma, S8636)</li>
	<li>12.5 ml HEPES solution 1 M (Sigma, H0887)</li>
	<li>5 ml N-2 Supplement (100X), liquid (Invitrogen, 17502-048)</li>
	<li>50 ml Horse Serum, Heat-Inactivated (Invitrogen, 26050-088)</li>
</ul>


Papain solution:
<ul>
	<li>Add 5 ml of dissection media into Worthington PAPAIN-022 vial (LK003178; final concentration, 20 U/ml) and incubate at 37&#176;C for 60 min.</li>
</ul>

<ol>
	<li>Decapitate rat pups aged P1 (typically 2 pups) following guidelines vetted and approved by national regulations and your local Institutional Animal Care and Use Committee.</li>
	<li>Remove skin and skull and place brain in Petri dish filled with cold dissection media.</li>
	<li>Removed meninges, dissect both hemispheres and dissect hippocampi.</li>
	<li>Chop the hippocampi into ~1X1 mm pieces and digest in papain solution at 37&#176;C for 11-13 min.</li>
	<li>Once the pieces of tissue have settled, remove papain carefully and add 5 ml of hippocampal medium to remove all traces of the enzyme. Repeat this step and resuspend in hippocampal medium (2 ml).</li>
	<li>Triturate the cells ~ 5 times until there are no clumps left, with three flame-polished pipettes of progressively smaller bores (1000 &#956;m, ~500 &#956;m and ~300 &#956;m).</li>
	<li>Once triturated, pass the cells through a cell strainer (pore size, 70 &#956;m, BD Bioscience). Count the cells in 10 &#956;l of suspension. Adjust the volume of hippocampal medium in order to have 400000 cells/ml.</li>
	<li>Aspirate the laminin off the coverslips, and before the covers can dry, plate 200 &#956;l of the cell suspension per coverslip.</li>
	<li>Leave them to attach for 60 min in the incubator, and then add 2 ml of hippocampal medium per well.</li>
	<li>The next day, aspirate old hippocampal medium to remove dead cells and debris and add 2 ml of pre-warmed fresh hippocampal medium.</li>
</ol>

Maintenance
Neurobasal media:
<ul>
	<li>500 ml neurobasal (Invitrogen, 12348-017)</li>
	<li>10 ml B27 serum free supplement (Invitrogen, 17504-044)</li>
	<li>5 ml Penicillin-Streptomycin&#160; (Invitrogen, 15140-122)</li>
	<li>1.25 ml L-glutamine (Invitrogen, 25030-149)</li>
</ul>
Feed the astrocyte-neuron cultures twice a week with neurobasal medium, starting four days after plating. Preincubate the media about 30 min in the incubator in a ventilated flask to equilibrate the temperature and CO2.
Part 2: CALCIUM IMAGING
Hippocampal recording buffer: 110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM D-glucose, 10 mM HEPES (All chemicals from Sigma) pH 7.4 (adjusted with NaOH).
Loading calcium indicator dye into astrocytes
<ol>
	<li>Place the culture in a well on a 6-well plate filled with 2 ml hippocampal recording buffer containing 2.5 &#956;M Fluo-4, AM (Invitrogen, F-14217) and 0.05% Pluronic&#174; F-127 20% solution in DMSO (Invitrogen, P-3000MP) and incubate at room temperature for 10-30 min.</li>
	<li>Remove Fluo-4 solution and wash coverslips 3 times with hippocampal recording buffer and incubate at room temperature for 30 min.</li>
	<li>Place the coverslip on the chamber, and clean the other side of coverslip with lens cleaning liquid.</li>
	<li>Put one small drop of immersion oil (Immersion Oil TYPE DF from Cargille) on the objective and put chamber (Open chamber for 25 mm round coverslip from Warner Instruments) on the stage of the microscope (Olympus IX71).</li>
	<li>Look at the cells using transmission light to see how the cells look, and get them into focus. Then illuminate the cells with 488 nm from a monochromator (Polychrome V from TILL Vision) and determine if the fluorescence signal of Fluo-4 is uniform and detectable in astrocytes.</li>
	<li>Use EPI and TIRF illumination to measure calcium in astrocytes.</li>
</ol>

TIRF microscopy
Briefly, we use an Olympus IX71 microscope equipped with an Andor IXON DV887DCS EMCCD camera.&#160; The control of excitation and image acquisition is achieved using TILLVision software.&#160; The beams of 454/488/515 nm Argon (100 mW) and 442 nm solid state (45 mW) lasers are combined and controlled with a TILL Polyline laser combiner, TIRF dual port condenser and acoustoptical tuneable filter and controller (AOTF; all from TILL Photonics) and fed into a Kineflex broad band fiber for entry into the TIRF condenser. We use an Olympus 60X 1.45 NA lens to achieve TIRF. The camera gain is adjusted for each astrocyte to provide the best signal to noise images. The background and principles of TIRF microscopy have been recently reviewed4, 5. Most of the optical components we use were purchased from TILL Photonics, which is now part of Agilent Technologies (http://www.till-photonics.com/). The TIR penetration depth can be calculated from the equations below.
<img alt="Equation" src="/files/ftp_upload/1142/equation.jpg" style="float:left;" />

d = penetration depth
n1 = refractive index of glass
n2 = refractive index of cell
a = angle of incidence
NAi = numerical aperture of incidence
 
In order to ensure the laser is aligned optimally for TIRF we find it useful to observe 100 nm fluorescent bead (Invitrogen, F8803). We present still frames and videos of beads with EPI and TIRF microscopy. When in TIRF, one observes a dramatic increase in signal-to-noise and the beads display Brownian diffusion. We find it useful to observe the behavior of 100 nm beads with TIRF microscopy on a regular basis (~once per week) to be sure that optimal TIRF occurs, rather than the compromised oblique illumination that would occur if the critical angle were not equal to &#945; (see Fig 1).
Application of G-protein coupled receptor agonists
Astrocytes express a variety of Gq-coupled receptors6, 7 including metabotropic glutamate receptors and P2Y receptors (agonist, ATP, ADP). Activation of these receptors leads to significant increases in intracellular calcium levels within astrocytes. For instance, one can readily observe intracellular calcium elevations during application of ATP (30 &#956;M) to astrocytes8-10. We use a fast solution switcher from Warner Instruments called the VC-77SP Fast-Step Perfusion System (http://www.warneronline.com/index.cfm). With this method solutions can be applied in less than ~10 ms.
<img alt="Figure 1" src="/files/ftp_upload/1142/fig1a.jpg" /> 
 
 
 
 
 
<img alt="" src="/files/ftp_upload/1142/fig1c.jpg" />
FIGURE 1. Cartoon and photographs of the imaging set up. A. Shows a photograph of the microscope mounted on an airtable, whereas (B) shows a photograph of the laser assembly, controllers and beam boxes. C. Shows a photograph of the microscope stage with the chamber mounted for imaging.&#160; On the left the fast perfusion device can be seen (along with the stepper motor and theta tubing). On the right the headstage of an Axopatch 200A amplifier is seen.&#160; The cartoon schematizes the light path in the set up and how TIRF is achieved. The laser is focused on the back focal plane of the 60X 1.45 NA objective lens and its position is adjusted off center so that it emerges into the immersion oil at the critical angle &#945;. At this point the beam suffers from total internal reflection and decays with a distance &#955; (see equation in main text) into the medium of lower refractive index. In this case this is the recording buffer surrounding the astrocytes and the astrocytes themselves. The result is optical excitation (and thus imaging) of molecules within ~100 nm of the plasma membrane. In the cartoon of the cell this is shown as green &#8220;excited&#8221; membrane receptors, whereas those within the cell or on the top surface of the cell are not excited. A full account of TIRF microscopy has been provided by Steyer and Almers4.
<img alt="Figure 2" src="/files/ftp_upload/1142/fig2.jpg" />
FIGURE 2. Images of 100 nm fluorescent beads acquired with EPI and TIRF microscopy. A. Shows EPI images of a field of view with several dozen 100 nm fluorescent beads. The red arrows point to beads that have settled onto the glass coverslip, whereas the blue arrowheads point to beads that are diffusing in water. B. Shows a TIRF image of the same field of view as shown in A. In this view only the adherent beads shown by red arrows are visible. This is because these had settled onto the glass coverlsip and were thus within the ~100 nm evanescent field. The beads shown in A by blue arrows are not within this region and are thus invisible in the TIRF images. The lower plots show 3D rendering of the images. It is clear that a large increase in signal-to-noise occurs for beads within the evanescent field when observed by TIRF microscopy. In fact for these images the signal-to-noise for EPI was 7.1 &#177; 0.6, whereas for TIRF it was 20 &#177; 0.7.
<img alt="Figure 3" src="/files/ftp_upload/1142/fig3.jpg" />
FIGURE 3. Images of astrocytes loaded with Fluo-4 calcium indicator dye acquired with EPI and TIRF microscopy.&#160; A. EPI images of a field of view with five astrocytes. B. A TIRF image of the same field of view shown in B. Note that the images in A and B are significantly different. This is because with TIRF illumination only the plasma membrane regions in close apposition to the glass coverslip are imaged.&#160; The lower panels show ATP-evoked intracellular calcium transients imaged with EPI and TIRF microscopy.

<ol>
	<li>Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+image+activation+of+transmitter-gated+P2X+receptors+in+vitro+and+in+vivo.">An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo.</a> Nature Methods. 5, 87-93 (2008).</li><li>Granseth, B., Odermatt, B., Royle, S. J., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+is+the+dominant+mechanism+of+vesicle+retrieval+at+hippocampal+synapses.">Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.</a> Neuron. 51, 773-786 (2006).</li><li>Nunez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+hippocampal+neurons+from+P0+newborn+rats.">Primary culture of hippocampal neurons from P0 newborn rats.</a> J Vis Exp. 19, (2008).</li><li>Steyer, J. A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+view+of+life+within+100+nm+of+the+plasma+membrane.">A real-time view of life within 100 nm of the plasma membrane.</a> Nat Rev Mol Cell Biol. 2, 268-275 (2001).</li><li>Jaiswal, J. K., Fix, M., Takano, T., Nedergaard, M., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+vesicle+fusion+from+lysis+to+monitor+calcium-triggered+lysosomal+exocytosis+in+astrocytes.">Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes.</a> Proc Natl Acad Sci U S A. 104, 14151-14156 (2007).</li><li>Araque, A., Carmignoto, G., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+signalling+between+astrocytes+and+neurons.">Dynamic signalling between astrocytes and neurons.</a> Annu Rev Physiol. 63, 795-813 (2001).</li><li>Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLIA:+listening+and+talking+to+the+synapse.">GLIA: listening and talking to the synapse.</a> Nat Rev Neurosci. 2, 185-193 (2001).</li><li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+excites+interneurons+and+astrocytes+to+increase+synaptic+inhibition+in+neuronal+networks.">ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks.</a> J Neurosci. 24, 8606-8620 (2004).</li><li>Bowser, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Lee, S. Y., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+glutamate+targets+NMDA+receptors.">Astrocytic glutamate targets NMDA receptors.</a> J Physiol. 581, 887-888 (2007).</li><li>Shigetomi, E., Bowser, D. N., Sofroniew, M. V., Khakh, B. 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It is well-established that astrocytes display intracellular calcium elevations. These occur spontaneously, can be triggered by neuronal activity or by application of agonists to activate receptors on the astrocyte surface11. One important and controversial issue is whether astrocyte intracellular calcium elevations can trigger the release of signaling molecules that activate receptors on neurons11, 12. This is controversial because there has been evidence for and against this view, as highlighted i...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
<td>Tool</td>
<td>VWR</td>
<td>48380-068</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
<td>&nbsp;</td>
<tr>
<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14155-063</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>51200-038</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>HEPES solution 1 M</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</td>
<td>&nbsp;</td>
<tr>
<td>PAPAIN-022</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LK003178</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal&#x2122; Medium (1X) Liquid without Phenol Red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>12348-017</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement (50X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine-200 mM (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-149</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainers</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M2670</td>
<td>&nbsp;</td>
<tr>
<td>HEPES free acid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
<tr>
<td>Fluo-4, AM 1 mM solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14217</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic&#174; F-127 20% solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil TYPE DF</td>
<td>Microscope</td>
<td>Cargille</td>
<td>16242</td>
<td>&nbsp;</td>
<tr>
<td>Open chamber for 25 mm round coverslips, 100 &mu;l volume</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0362 (RC-21BDW)</td>
<td>&nbsp;</td>
<tr>
<td>P-2 platform for Series 20 chambers, non-heater</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0278 (P-2)</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres carboxylate-modified microspheres, 0.1 &mu;m, yellow-green fluorescent (505/515) 2% solids</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F8803</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

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, ...

measuring-near-plasma-membrane-global-intracellular-calcium-dynamics

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JoVE Journal

Biology

13.0K Views.  David Geffen School of Medicine, University of California, Los Angeles. We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Video: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

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 Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

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 ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...