This work was supported by the Ministry of Education Science and Technological Development Republic of Serbia Contract No. 451-03-9/2021-14/ 200178, the FENS - NENS Education and Training Cluster project &#34;Trilateral Course on Glia in Neuroinflammation&#34;, and the EC H2020 MSCA RISE grant #778405. We thank Marija Ad&#382;i&#263; and Mina Peri&#263; for supplying the immunohistochemistry images and Danijela Batavelji&#263; for help with paper writing.

All experiments were performed in accordance with the EU directives on the protection of animals for scientific purposes and with permission from the Ethical Commission of the Faculty of Biology, University of Belgrade (approval number EK-BF-2016/08). Regarding patient material (sera for IgGs), it was collected for routine clinical examination with informed patient&#39;s consent in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The protocol was approved by the Ethics committee of the Clinical Center of Serbia (No. 850/6).
1. Primary cell culture preparation
<ol>
	<li>Brain tissue isolation 
	NOTE: Isolation should be performed on ice, using ice cold solutions.
	<ol>
		<li>Use newborn pups 1-3 days old for primary neonatal cell culture33. 
		NOTE: For the study presented here, Sprague Dawley hSOD1G93A and non-transgenic rats were used. For genotyping, rat tails were used for later DNA extraction and PCR.</li>
		<li>Sprinkle the pup&#39;s head with 70% ethanol and immediately decapitate it fast using scissors.</li>
		<li>Cut open the skin using small-angled scissors to expose the skull. Open the skull by making a cut from the foramen magnum toward the orbits. Next, make a perpendicular midline cut. Remove the brain from the skull and place it in a Petri dish containing phosphate-buffered saline (PBS). 
		NOTE: Clean the tools between pups to prevent cross-contamination between transgenic and non-transgenic pups. Isolation of the cortices from the rest of the brain should be done under a stereomicroscope.</li>
		<li>Using the tip of the forceps, tear the connections between both hemispheres. Then, separate the hemispheres with curved forceps by gently pushing a hemisphere from the center to the side.</li>
		<li>Remove the meninges by carefully tearing them with straight and curved forceps.</li>
		<li>Remove the hippocampus by pinching it with a curved forceps. Discard the hippocampus or use it for another cell culture preparation. 
		NOTE: Further steps should be performed under the laminar flow hood to ensure sterile conditions.</li>
	</ol>
	</li>
	<li>Tissue homogenization and dissociation
	<ol>
		<li>Transfer one cortex into a 15 mL tube filled with 3 mL of cold PBS. Pipet the suspension up and down with a 1 mL tip, completing 10-15 strokes until the suspension becomes homogeneous. 
		NOTE: Be very cautious not to produce bubbles in this process.</li>
		<li>Centrifuge at 500 &#215; g for 5 min. Remove the supernatant and resuspend the pellet in 3 mL of cold PBS by pipetting it up and down with a 1 mL tip. Repeat the centrifugation step. 
		NOTE: All solutions used from this point should be prewarmed to 37 &#176;C.</li>
		<li>Discard the supernatant and resuspend the pellet in 2 mL of complete Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Transfer the homogenate to a 2 mL tube.</li>
		<li>Pass the homogenate through 21 G and 23 G needles (three times each) to make a suspension of single cells. 
		NOTE: Be very cautious not to produce bubbles in this process.</li>
	</ol>
	</li>
	<li>Pour the cell suspension prepared from one cortex into a 60 mm diameter Petri dish or a T25 flask (with the surface pretreated with a polypeptide coating for the growth of adherent cells) containing 3 mL of complete DMEM. Lightly shake the Petri dish so that the suspension is uniformly distributed.</li>
	<li>Grow the cells in the incubator in a humidified atmosphere of 5% CO2/95% air at 37 &#176;C.</li>
	<li>Change the medium 48 h after isolation and replace the medium every 3 days.</li>
	<li>To promote astrocyte growth, when cells reach 70%-80% confluency, wash them with prewarmed PBS to remove the loosely attached glial cells and traces of FBS in the medium.
	<ol>
		<li>Trypsinize the underlying layer of astrocytes by adding 1 mL of prewarmed trypsin solution (0.25% trypsin, 0.02% EDTA in PBS, sterile-filtered).</li>
		<li>Place the dish in the incubator at 37 &#176;C for 2-5 min.</li>
		<li>Check the cells under the microscope. When they start to detach, add 4 mL of complete DMEM.</li>
		<li>Collect the cell suspension and transfer it to a 15 mL tube. Centrifuge at 500 &#215; g for 5 min.</li>
		<li>Discard the supernatant and resuspend the pellet in 1 mL of complete medium. Count the cells using a hemocytometer.</li>
		<li>Replate the cells at a density of 104 cells/cm2 in 5 mL of fresh complete DMEM.</li>
	</ol>
	</li>
	<li>Change the medium every 3 days. To minimize the presence of other glial cell types, after astrocytes reach 50% confluence and prior to each media replacement, wash the cells with complete DMEM.
	<ol>
		<li>Aspirate the supernatant medium with a 1 mL pipette and gently dispense it onto the layer of cells several times. Ensure that the whole surface of the cell layer is covered during this washing step.</li>
	</ol>
	</li>
	<li>Once the cells reach 80% confluence (usually after 14 days), repeat the trypsinization and the collection of astrocytes as described in step 1.6.</li>
	<li>Seed 5 &#215; 103 of astrocytes on a 7 mm circular glass coverslip coated with poly-L-lysine (50 &#181;g/mL). Use them in experiments after 48 h.</li>
	<li>To promote microglia, after step 1.7, allow the glial cells to reach a confluent layer34. When microglial cells appear on top of the astrocyte layer (after 10-15 days, recognized by their smaller, more oval bodies and shorter processes), shake the Petri dish on an orbital shaker for 2 h at 220 rpm.</li>
	<li>Dispersing and seeding microglial cells
	<ol>
		<li>Lightly wash the detached and loosely attached cells by aspirating the supernatant medium with a 1 mL pipette tip and gently dispense it onto the layer of cells several times. Be sure to cover the whole surface of the cell layer during this washing step, collect the medium with the detached cells, and transfer it to a 15 mL tube.</li>
		<li>Centrifuge at 500 &#215; g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of medium.</li>
		<li>Pass the cell suspension through a 21 G needle to obtain a single-cell suspension. 
		NOTE: Most published methods use trypsin or papain to dissociate the tissue; however, 21 G needles have the advantage of preventing the overdigestion of cells, allowing for a gentler dissociation.</li>
		<li>Count the cells using a hemocytometer. Seed 5 &#215; 103 microglial cells on a 7 mm circular glass coverslip coated with poly-L-lysine (50 &#181;g/mL). Use them in experiments after 48 h. 
		&#8203;NOTE: It is difficult to obtain a pure microglial culture by shaking, since the oligodendrocyte precursor cells and astrocytes may still be present in a variable number depending on the experimenter&#39;s experience. The immunocytochemical protocol used to estimate the presence of different cell populations in glial cultures has been described elsewhere35.</li>
	</ol>
	</li>
</ol>
2. Immunocytochemistry
<ol>
	<li>Rinse the cells plated on coverslips in PBS, 2 x 1 min.</li>
	<li>Fix the cells in 4% paraformaldehyde for 20 min at room temperature (RT).</li>
	<li>Wash in PBS 3 x 5 min.</li>
	<li>Incubate in a blocking solution containing 10% normal donkey serum (NDS)/1% bovine serum albumin (BSA)/0.1% Triton X-100 for 45 min at RT.
	<ol>
		<li>Add 50 &#181;L of a blocking solution per 10 mm diameter coverslip.</li>
	</ol>
	</li>
	<li>Prepare primary antibodies in 1% NDS/1% BSA/0.1% TritonX-100 in the following dilutions: mouse anti-GFAP 1:300, goat anti-Iba1 1:500. Incubate the cells with primary antibodies overnight at +4 &#176;C.</li>
	<li>Rinse in PBS, 3 x 10 min.</li>
	<li>Incubate with the secondary antibodies conjugated with a fluorophore: donkey anti-mouse (excitation 488 nm [1:200)) or donkey anti-goat (excited at 647 nm [1:200)). Incubate the cells for 2 h at RT in the dark.</li>
	<li>Wash in PBS, 7 x 5 min.</li>
	<li>Incubate the cells with 4&#39;,6-diamidino-2-phenylindole (DAPI, 1:4,000) for 10 min at RT.</li>
	<li>Wash in PBS 5 x 5 min.</li>
	<li>Mount the coverslips on microscope slides using a mounting solution; use one drop of it per coverslip. 
	NOTE: Antibody dilutions may vary depending on the producer. Users must determine optimal dilutions for their experiments.</li>
</ol>
3. Time-lapse video imaging
NOTE: Solutions containing the fluorescent dye should be protected from direct light. Before starting the imaging experiment, make sure that the glass coverslip does not move when turning on the perfusion.
<ol>
	<li>Preparation of solutions and cells
	<ol>
		<li>Prepare extracellular solution (ECS) for imaging: 140 mM NaCl, 5 mM KCl, 2 mM CaCal2, 2 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES. Adjust the pH to 7.4. Ensure that the osmolarity is 280-300 mOsm/kg and prepare ECS without Ca2+: 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, and 0.1 mM EGTA.</li>
		<li>Prepare the test solutions: 100 &#181;M ATP dissolved in ECS, 1 &#181;M Thg in ECS without Ca2+, and 0.1 mg/mL ALS IgG in ECS8.</li>
		<li>Transfer one coverslip to a dish with ECS to wash out the complete DMEM. Prepare the dye loading solution by diluting 1 mM stock solution of Fluo-4 AM with ECS to the final concentration of 5 &#181;M Fluo-4 AM.
		<ol>
			<li>Place the coverslip with cells in the dye loading solution for 30 min at RT in the dark. Wash the cells in ECS for 20 min.</li>
		</ol>
		</li>
		<li>If cells are not adequately loaded (i.e., if the basal level of fluorescence is too low-&#60;5% of the dynamic range of the camera with exposure time greater than 400 ms), increase the loading time to up to 45 min to 1 h at 37 &#176;C. Alternatively, mix the Fluo-4 AM stock solution with an equal volume of 20% (w/v) detergent (Pluronic) in DMSO before diluting in ECS, making the final Pluronic concentration ~0.02%. 
		NOTE: The experimental examples of this study were performed with human IgG isolated from ALS patients&#39; blood sera as described previously4,5,11. Each experiment was performed with IgG samples of a single patient.</li>
	</ol>
	</li>
	<li>Video imaging 
	NOTE:&#160;In this protocol, the Video Imaging System was combined with a xenon short arc lamp, a polychromator system, and an inverted epifluorescence microscope equipped with water, glycerin, and oil immersion objective. Timelapse imaging was performed using a Digital Camera System.
	<ol>
		<li>Switch on the components of the imaging setup 15 min before the experiment to allow the system to reach the working temperature.</li>
		<li>Place the coverslip in the recording chamber with 1 mL of working solution (ECS or ECS without Ca2+).</li>
		<li>To choose the correct imaging protocol, open the imaging software and in the Acquisition panel, choose the filter pairs for Fluo4-AM with an excitation wavelength at 480 nm, a dichroic mirror at 505 nm, and an emission wavelength at 535 nm.</li>
		<li>Choose the field of view taking care to have a consistent number of cells throughout the experiment. Adjust the exposure time and detector gain appropriately, in such a manner that the signal is not saturated. Choose the pseudocolor mode for acquisition. For more detailed instructions, refer to the manual provided by the manufacturer.</li>
		<li>Adjust the sampling rate to 1 Hz (1 frame per second).</li>
		<li>Start the imaging by acquiring the basal level of fluorescence for 3-5 min for the baseline determination (F0). Ensure a constant flow of the working solution.</li>
		<li>Stop the flow of the working solution and switch to the test solution for the desired length of time (also see time bars in examples of Figure 2 and Figure 3). Between each test solution, wash the cells with a constant flow of the working solution for 3-5 min.</li>
		<li>Keep the solution volume in the recording chamber at ~1 mL by arranging a constant suction from the top of the solution (see Figure 2E). 
		&#8203;NOTE: To apply the treatment directly to the imaged cells, a customized delivery system made of a glass pipette (0.8 mm inner diameter) was positioned ~350 &#181;m away and ~1 mm above the cells, at an angle of 45&#176; combined with the solution exchange system containing pinch valves and an electronic valve controller (see Figure 2E).</li>
	</ol>
	</li>
</ol>
4. Data analysis
<ol>
	<li>Define a region of interest (ROI) that corresponds to the individual cell.
	<ol>
		<li>Choose the frame with the highest signal intensity (i.e., from the application of ATP) and encircle one cell by using the application Polygonal Tool. Repeat for all the cells in the acquired field. Choose five ROIs in the background using the Circle Tool.</li>
		<li>To measure the mean signal intensity of a single cell and the background for each time frame, select all ROIs and use the Multi Measure command in ROI Manager in ImageJ or any equivalent command in commercial software (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Export the mean signal intensity values of the ROIs as a spreadsheet.</li>
	<li>Average five background ROIs and subtract the averaged background of each frame from the mean ROI intensity of the frame acquired at the same time.</li>
	<li>To normalize the obtained data to the baseline signal, use equation (1): 
	&#916;F/F0 = (F - F0)/F0&#160; &#160;(1) 
	where F is the signal intensity of each time frame after background subtraction, and F0 is the baseline Fluo-4 fluorescence. 
	NOTE: A custom-made code was used in this study.</li>
	<li>To analyze calcium activity, determine several parameters4: the amplitude of the calcium peak (&#916;F/F0), the integrated change (time integral, surface under the response recording) of the calcium transient (&#916;F/F0 &#215; s), time-to-peak-elapsed time from the onset of stimulation to the maximum of calcium transient (s), rise time-time elapsed from the onset of the stimulation to the value of &#916;F/F0 that reaches 50%-80% of the maximal amplitude (s), half-width-full width of a transient at half-maximal amplitude (s), frequency of responses if they are repetitive (Hz).</li>
</ol>

The authors have no conflicts of interest to declare.

This paper presents the method of primary cell culturing as a fast and &#34;on the budget&#34; tool for studying different aspects of cell (patho)physiology such as ALS in the rat hSOD1G93A model. The technique is thus suitable for studies at the single-cell level that can be extrapolated and further investigated at a higher level of organization (i.e., in tissue slices or in a live animal). Cell culturing as a technique, however, has a few caveats. It is most critical to do the brain tissue isolation and the ...

Cell culture techniques have given rise to numerous advancements in diverse fields of cellular neurophysiology in health and disease. Particularly, primary cell cultures, freshly isolated from the neuronal tissue of a lab animal, allow the experimenter to closely study the behavior of diverse cells in different biochemical media and physiological setups. Using different fluorescent physiological indicators such as the Ca2+-sensitive dyes in combination with time-lapse video microscopy provides better insight into the cellular biophysical and biochemical processes in real time.
ALS is a devastating neurodegenerative disease that affects upper and lower motor neurons1. The disease has a complex pathogenesis of the familial type but mostly of the sporadic form (90% of cases)2. It is well known that non-cell autonomous mechanisms contribute to ALS pathophysiology, primarily due to the essential role of glial cells3. ALS is also well characterized as a neuroinflammatory disease with involvement of humoral and cellular factors of inflammation.
Immunoglobulin G is widely used as a molecular marker in ALS and other neurodegenerative diseases. Studying the serum level of this marker can indicate the presence and stage of neuroinflammation in the disease4,5,6, while its presence in the cerebrospinal fluid can indicate a breach of the blood brain barrier7. IgGs were also identified as deposits in the spinal cord motor neurons of ALS patients7. Nevertheless, this approach has shown some inconsistencies in the correlation of the level of IgGs with the stage and characteristics of the disease6.
IgG isolated from the sera of ALS patients (ALS IgG) can induce a calcium response in naive astrocytes8 and glutamate release in neurons, pointing to an excitotoxic effect-a hallmark of ALS pathology9. However, studies on the hSOD1G93A ALS rat model (containing multiple copies of the human SOD1 mutation10) showed a number of markers of oxidative stress in cultured neuroglial cells11, tissues12,13,14, or live animals13. It is noteworthy that the astrocytes cultured from the ALS rat model were more prone to oxidative stress induced by peroxide than the astroglia from non-transgenic littermates11.
Microglial cells in culture are affected by ALS IgG in a less apparent way. Namely, a BV-2 microglial cell line displayed a rise in the signal from fluorescent markers of oxidative stress in response to the application of only 4/11 ALS IgG patient samples15. It is well known that microglia participate in many neuroinflammatory pathologies, adding to oxidative stress and late progression phase in the non-cell autonomous mechanism of ALS16,17. Nevertheless, the data with ALS IgGs indicated that these cells may not be as reactive as astrocytes to these humoral factors of ALS inflammation. Several studies have been conducted with primary astrocytes from ALS murine models, not only in pups but also in symptomatic animals, either on the brain or on the spinal cord18,19,20,21. This is also true for microglial primary cultures, although to a lesser extent than astrocytes and mostly from brain regions at the embryonic stage22,23,24.
We use time-lapse video imaging of Ca2+ on cells in culture primarily as a means to follow intracellular transients of this ion as a physiological marker of excitotoxicity. Thus, by biophysical characterization of these transients (amplitude, area under transient, rise-time, frequency) the researcher can obtain experimental diagnostic parameters from diverse cellular models of neurodegeneration. This technique thus offers an advantage of a quantitative physiological assessment of IgGs as disease biomarkers. There is a large body of literature on the role of IgGs and Ca2+ in the induction of ALS. Most of these studies were performed by inducing ALS by injecting patient IgGs into experimental animals25,26,27,28,29, which then showed intracellular Ca2+ elevation and IgG depositions. A line of studies explored the effect of ALS IgGs on the motor synapse in vitro30,31,32. In the above context, the technique presented here puts the focus on the glial cells as important players in the non-cell autonomous mechanism of ALS and quantifies their potential excitotoxic response to IgGs as humoral factors of neuroinflammation. This approach may have a wider application in testing other humoral factors such as whole sera, CSF, or cytokines in different cell culture systems and in cellular models of general inflammation.
This paper describes how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to further use these cells to study ALS pathophysiology with patient sera-derived IgG. Protocols are detailed for the dye-loading of cultured cells (Figure 1) and the recordings of Ca2+ changes in time-lapse video imaging experiments. Examples of video recordings will show how glial cells react to ALS IgG as compared to ATP, the latter activating purinergic membrane receptors. Shown for the first time is an example on how astrocytes isolated from the hSOD1G93A ALS rat brain react with a more pronounced Ca2+ response to ALS IgG compared to non-transgenic controls and how to relate this process to the differences in Ca2+ store operation. Also shown is an example of calcium imaging in microglial cells acutely challenged with ALS IgG, with only a modest response of intracellular calcium.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL tube</td>
			<td>Sarstedt, Germany</td>
			<td>62 554 502</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL tube</td>
			<td>Sarstedt, Germany</td>
			<td>72.691</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>Nipro, Japan</td>
			<td>HN-2138-ET</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>Nipro, Japan</td>
			<td>HN-2338-ET</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Nipro, Japan</td>
			<td>SY3-5SC-EC</td>
			<td></td>
		</tr>
		<tr>
			<td>6 mm circular glass coverslip</td>
			<td>Menzel Glasser, Germany</td>
			<td>630-2113</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>ThermoFisher Sientific, USA</td>
			<td>130181</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>A9062</td>
			<td></td>
		</tr>
		<tr>
			<td>AxioObserver A1</td>
			<td>Carl Zeiss, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>B6917</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>2110</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>158968</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey-anti goat AlexaFluor 647 IgG antibody</td>
			<td>Invitrogen, USA</td>
			<td>A-21447</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey-anti mouse AlexaFluor 488 IgG antibody</td>
			<td>Invitrogen, USA</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>EDS-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8221;evolve&#8221;-EM 512 Digital Camera System</td>
			<td>Photometrics, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji ImageJ Software</td>
			<td>Open source under the GNU General Public Licence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FITC filter set</td>
			<td>Chroma Technology Inc., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Molecular Probes, USA</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Iba1</td>
			<td>Fujifilm Wako Chemicals, USA</td>
			<td>011-27991</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Biowest, France</td>
			<td>P5455</td>
			<td></td>
		</tr>
		<tr>
			<td>HighSpeed Solution Exchange System</td>
			<td>ALA Scientific Instruments, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert GmbH + Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab software</td>
			<td>Math Works, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-GFAP</td>
			<td>Merck Millipore, USA</td>
			<td>MAB360</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 40-88</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>324590</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin and Streptomycin</td>
			<td>ThermoFisher Sientific, USA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carlo Erba Reagents, Spain</td>
			<td>471686</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker DELFIA PlateShake</td>
			<td>PerkinElmer Life Sciencies, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S3817</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic heptahydrate</td>
			<td>Carl ROTH GmbH</td>
			<td>X987.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Thapsigargine</td>
			<td>Tocris Bioscience, UK</td>
			<td>1138</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X - 100</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Vapro Vapor Pressure Osmometer 5520</td>
			<td>Wescor, ELITechGroup Inc., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ViiFluor Imaging System</td>
			<td>Visitron System Gmbh, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VisiChrome Polychromator System</td>
			<td>Visitron System Gmbh, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VisiView high performance setup</td>
			<td>Visitron System Gmbh, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xenon Short Arc lamp</td>
			<td>Ushio, Japan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

primary cultures of rat astrocytes and microglia and their use in the study of amyotrophic lateral sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

Characterization of different glial cell types in culture 
It usually takes 15-21 days to produce astrocytes for experiments, while microglial cells take 10-15 days to grow. Immunostaining was performed to assess the cell purity of the culture. Figure 1 shows the expression of double labeling of the astrocytic marker GFAP and the microglial marker Iba1 in respective cultures.
Calcium imaging is known to reveal the differences in cell physiolo...

Watch this Scientific Journal Video about Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis at JoVE.com

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

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3.2K Views.  University of Belgrade Faculty of Biology. We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

Video: Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALS&#160;models and any C. elegans strain that exhibits altered motility.

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...