This work is supported by FAPESP (Grant 25681-3/2017 to AS), CAPES (Post-Doctoral fellowship PNPD/INCT-HSM to A.F. and Pre-Doctoral fellowship to N.D.M.) and FAEPA. G.M.A. holds a Master&#8217;s fellowship from FAPESP (MS 2018/06614-4). N.G.C. holds a CNPq Research Fellowship. We thank the patients and their families for donating the resected tissues for this study. We would like to acknowledge the support of residents, nurses, technicians, and the CIREP team, from the Clinical Hospital at the Ribeir&#227;o Preto Medical School, University of S&#227;o Paulo, who helped in various stages of the process.

Live adult brain tissues were obtained from patients undergoing resective neurosurgery for the treatment of pharmacoresistant temporal lobe epilepsy (Figure 1A). All procedures were approved by the Ethics Committee from the Clinics Hospital at the Ribeir&#227;o Preto Medical School (17578/2015), and patients (or their legal responsible person) agreed and signed the informed consent terms. Collection of the tissue was done by the neurosurgery team at the Epilepsy Surgery Center (CIREP - Clinics Hospital at the Ribeir&#227;o Preto Medical School, University of S&#227;o Paulo, Brazil).
1. Sterilization of materials
NOTE: All material and solutions must be sterilized prior to use.
<ol>
	<li>Sterilize all surgical tools and vibratome slicing material (knife holder, specimen disk, buffer tray) in a dry sterilizing oven for 4 h at 180 &#176;C.</li>
	<li>Sterilize temperature-sensitive material or equipment by UV or gamma irradiation.</li>
	<li>Sterilize media and solutions by autoclavation or filtration through 0.22 &#181;m pore membranes.</li>
</ol>
2. Preparation of solutions
<ol>
	<li>Prepare 15-20 mL of transport solution: 50% v/v Hanks&#39;&#160;balanced salt solution (HBSS) pH 7.4, 50% v/v basal medium for maintenance of post-natal and adult brain neurons (Table of Materials), supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 3 mg/mL glucose, and 33 &#181;g/mL gentamicin. 
	NOTE: Transport solution must be refrigerated and oxygenated (bubbling with carbogen gas) for at least 20 min prior to sample collection.</li>
	<li>Prepare 300 mL of slicing solution (HBSS supplemented with 10 mM HEPES and 3 mg/mL glucose) and cool it down in a freezer to the point of initial crystal formation.</li>
	<li>Prepare 20 mL of culture medium: basal medium for maintenance of post-natal and adult brain neurons (Table of Materials) supplemented with 1% L-glutamine derivative (Table of Materials), 2% supplement for neural culture (Table of Materials), 1% penicillin/streptomycin, and 0.25 &#181;g/mL amphotericin B.</li>
</ol>
3. Setting up the slicing apparatus
NOTE: This protocol is ideally performed with the assistance of a colleague due to the logistics of sample collection in the surgical room.
<ol>
	<li>In a bucket of salt-added ice, let the slicing solution rest under carbogen mixture bubbling (95% O2, 5% CO2) for at least 20 min prior to use.</li>
	<li>Prepare a block of 3% agarose (approximately 2 cm x 2 cm x 2 cm) and superglue it to the vibratome specimen disk in order to create additional mechanical support to the tissue sample during slicing (Figure 1E).</li>
	<li>Set the vibratome for slicing: section thickness of 200 &#181;m, frequency of vibration of 100 Hz, and speed of slicing between 0.5-1.0&#160;mm/s.</li>
	<li>Lock the vibratome buffer tray to the vibratome base and add ice to keep it refrigerated prior to receiving the slicing solution and the sample, and throughout the slicing procedure.</li>
</ol>
4. Sample collection
NOTE: In this protocol, human neocortical tissue was collected in the surgical room and transported to the laboratory.
CAUTION: When dealing with human samples, follow the appropriate safety protocols established by the Institution.
<ol>
	<li>Set up the transport apparatus (Figure 1C) that consists of: a portable gas cylinder with carbogen mixture connected to a pressure/flux valve that controls the gas output connected to a silicon tubing that connects gas output to the transport vessel; a transport vessel, usually a 50 mL conical centrifuge tube with perforated lid for gas input, containing the transport solution; and ice for sample cooling during transport.</li>
	<li>Collect and transport the specimen (Figure 1B) immediately to the lab. Submerse the specimen in cold transport solution (constantly bubbled with carbogen mixture).</li>
</ol>
5. Slicing
<ol>
	<li>Transfer the specimen to a Petri dish (100 mm x 20 mm) containing slicing solution and, with fine surgical tools, carefully remove as much as possible of the remaining meninges in the sample (Figure 1D).</li>
	<li>Choose the best specimen orientation for producing slices with the particular characteristics of the experimental design, and with a no. 24 scalpel blade, trim a flat surface to be the base glued to the specimen disk.</li>
	<li>Using a disposable plastic spoon and delicate paintbrushes, collect the fragment from the Petri dish and dry excess solution using filter paper (dry by capillarity and avoid touching the tissue fragment with paper).</li>
	<li>Using superglue, attach the tissue to the vibratome specimen disk until it is firmly adhered to the disk and in contact with the agarose block (Figure 1E).</li>
	<li>Place the vibratome specimen disk (with tissue properly attached) in the vibratome buffer tray filled with slicing solution that must be bubbling during the whole process.</li>
	<li>Lock the knife holder in place with the razor blade firmly fixed.</li>
	<li>The slicing solution must cover both the specimen and the blade, only then start slicing (Figure 1F).</li>
	<li>Cut the specimen into 200 &#181;m slices. 
	NOTE: Although some vibratomes cut the specimens automatically, the close observation and minor adjustments in slicing speed during the process may help producing better slices. Discard initial irregular slices.</li>
	<li>Transfer the slices from the buffer tray to a Petri dish with slicing solution and trim loose edges and excess white mater to a proportion of around 70% cortex/30% white matter.</li>
</ol>
6. Culture
NOTE: Perform this step in a laminar flow cabinet under sterile environment.
<ol>
	<li>Add 600 &#181;L of culture medium per well (in a 24 well plate) and incubate for at least 20 min at 36 &#176;C and 5% CO2 prior to plating the slices.</li>
	<li>Plate one slice per well using a paintbrush (Figure 1G).</li>
	<li>If there are any unused wells in the plate, fill them with 400 &#181;L of sterile water.</li>
	<li>Incubate the plate at 36 &#176;C, 5% CO2. 
	NOTE: First medium replacement must be done in between 8-16 h after plating depending on the size of the slice.</li>
	<li>Supplement 10 mL of the previously prepared culture medium with 50 ng/mL brain derived neurotrophic factor (BDNF). 
	NOTE: During the first 8-16 h, the slices are incubated in 600 &#181;L of medium to avoid nutrient deprivation and acidification, since medium consumption in this phase is accelerated. From the next step, the volume of medium per well is adjusted to 400 &#181;L.</li>
	<li>Remove 333 &#181;L of the conditioned medium from each well and add 133 &#181;L of fresh BDNF-supplemented medium.</li>
	<li>Repeat the process of medium replacement every 24 h by replacing one-third of the conditioned medium with fresh BDNF-supplemented medium.</li>
</ol>
7. Evaluating health, morphology, and function in cultured slices
NOTE: To induce cell death for the purpose of illustration in the representative results, some slices were submitted to a 24 h treatment with the oxidative stress inducer H2O2. Steps 7.1.2 and 7.1.3 describe use of H2O2 to induce cell death.
<ol>
	<li>Cell viability 
	NOTE: A simple, straightforward method to evaluate neurotoxic/neuroprotection in challenged slice cultures is the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay36, which measures the percentage of metabolic active cells under normal conditions compared to treated samples.
	<ol>
		<li>In the laminar flow cabinet, replace one-third of the medium with fresh medium.</li>
		<li>From a 30% H2O2 stock solution, add a volume of H2O2 per well to reach the intended final concentration (30 mM or 300 mM). 
		NOTE: H2O2 was added after the daily change of the medium to guarantee the proper supply of fresh nutrients prior to the challenge.</li>
		<li>Incubate the plate for 24 h at 36 &#176;C, 5% CO2. 
		NOTE: After H2O2 treatment (or other toxic stimulus of interest), the following steps describe cell viability determination by the MTT assay.</li>
		<li>Add 40 &#181;L of MTT solution to a final concentration of 0.5 mg/mL to each well in the plate.</li>
		<li>Incubate the plate at 36 &#176;C, 5% CO2 for 3 h.</li>
		<li>Wash the slices with phosphate-buffered saline (PBS) and transfer to a microtube.</li>
		<li>Carefully remove any remaining solution by pipetting.</li>
		<li>Weigh the microtubes containing the slices to determine the mass of each slice (this is key for normalizing the absorbance readings obtained). 
		NOTE: If needed, samples can be frozen (-20 &#176;C) at this stage for later processing.</li>
		<li>Homogenize the slices in 200 &#181;L of isopropanol/HCl using a motorized pestle.</li>
		<li>Centrifuge at room temperature (RT) for 2 min at 2600 x g.</li>
		<li>Collect the supernatant and measure the absorbance at 540 nm.</li>
	</ol>
	</li>
	<li>KCl-induced neuronal depolarization 
	NOTE: Phosphorylation of the mitogen activated protein kinase (MAPK) signaling cascade protein ERK, followed by western blotting, can be used for the quantification of the neuronal response to KCl-induced depolarization37 .
	<ol>
		<li>At the flow cabinet, replace the culture medium by 300 &#181;L of HBSS previously equilibrated to 36 &#176;C.</li>
		<li>Replace the HBSS with 300 &#181;L of fresh HBSS previously equilibrated to 36 &#176;C.</li>
		<li>Incubate the plate at 36 &#176;C, 5% CO2 for 15 min.</li>
		<li>Replace the HBSS with either the fresh HBSS or with 80 mM KCl depolarizing solution (both at 36 &#176;C) and incubate at 36 &#176;C, 5% CO2 for 15 min.</li>
		<li>Transfer the slices from the plate to microtubes. At this step, slices can be stored at -20 &#176;C for later processing.</li>
		<li>Prepare tissue extracts in 150 &#181;L of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% nonionic surfactant; 0.1% sodium dodecyl sulfate; pH 7.5). Centrifuge extracts at 4 &#176;C for 10 min at 16000 x g, collect supernatant, and determine total protein concentration using the Bradford method.</li>
		<li>Load 30 &#181;g of total protein onto a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).</li>
		<li>After electrophoresis, transfer the gel content to a nitrocellulose membrane.</li>
		<li>After blocking the membrane with 5% non-fat dry milk in TBS plus 0.1% Tween, incubate with mouse anti-pERK (1:1,000) for 16 h at 4 &#176;C. After washing, incubate for 2 h at RT with rabbit anti-ERK1/2 (1:1,000).</li>
		<li>Incubate with the appropriate HRP-conjugated secondary antibody at RT for 1 h.</li>
		<li>Reveal with the preferred HRP substrate.</li>
	</ol>
	</li>
	<li>Morphological evaluation 
	NOTE: In addition to cell survival and functional evaluations, it is important to analyze tissue morphology. Be aware that resectioning the cultured slice is an important step to producing as much high-quality material as possible for morphological analysis.
	<ol>
		<li>Fixing, cryoprotecting and resectioning the slices
		<ol>
			<li>Transfer the slices from the wells with culture medium to a new 24 well plate containing PBS.</li>
			<li>Remove the PBS and add 1 mL of 4% paraformaldehyde (PFA). It is important that slices be kept flat prior to adding PFA. Incubate overnight at 4 &#176;C.</li>
			<li>Carefully remove the PFA solution and add 1 mL of 30% sucrose solution. Incubate for 48 h at 4 &#176;C.</li>
			<li>Set the freezing microtome to -40 &#176;C.</li>
			<li>Prepare a sucrose base on the microtome stage where the slices should be placed (Figure 2A). Let it freeze completely and carefully cut some of the frozen sucrose to produce a flat surface on which the slice will be placed.</li>
			<li>Place each slice over a stretched plastic film and use a paintbrush to flat the tissue.</li>
			<li>Transfer the stretched slice to the frozen sucrose base in one single move. 
			NOTE: It is not possible to move the slice once it is over the frozen sucrose base. Perform this transfer step carefully.</li>
			<li>Let the slice rest for 5-10 min for proper freezing.</li>
			<li>Cut the slice into 30 &#181;m sections.</li>
			<li>Transfer the 30 &#181;m sections to a Petri dish containing PBS.</li>
			<li>Proceed to the histology protocol more adequate to the experimental design. 
			NOTE: The 30 &#181;m sections can be readily used for free-floating immunohistochemistry, mounted onto microscopy slides for further histology or stored in antifreeze solution at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Immunohistochemistry 
		NOTE: Immunohistochemistry and immunofluorescence standard protocols vary among labs. For a detailed version of the protocol used here, refer to Horta et al.38. Primary antibodies used for immunostainings presented in Figure 2 include neuronal nuclei (NeuN), healthy mature neuron marker, glial fibrillary acidic protein (GFAP), astorcytes marker, ionized calcium binding adapter (Iba-1), and microglia marker.
		<ol>
			<li>Incubate the slices in blocking solution (e.g., 2% normal donkey serum in PBS) for 40 min.</li>
			<li>Incubate overnight with primary antibody under mild agitation at 4 &#176;C.</li>
			<li>After washing with PBS, incubate for 120 min with biotinylated secondary antibody under mild agitation at RT.</li>
			<li>After washing with PBS, incubate at RT for 120 min with avidin-peroxidase conjugate (Table of Materials).</li>
			<li>Reveal with DAB + 0.04% nickel ammonium.</li>
			<li>Mount the stained sections on gelatin coated microscopy slides and let them air dry. Dehydrate in ethanol, diaphanize in xylene, finish with mounting medium (Table of Materials), cover with a coverslip, and image.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This protocol for producing free-floating, short-term slice cultures is an alternative method for culturing adult human neocortical slices. Such a protocol for slice cultures may be amenable for studies on (but not restricted to) optogenetics1,44,45, electrophysiology2,3,4,5, short-term plasticity<sup ...

The use of human samples in research is unequivocally a great option to study human brain pathologies, and modern techniques have opened new ways for robust and ethical experimentation using patient-derived tissue. Methods like organotypic/slice cultures prepared from adult human brain have been increasingly used in paradigms such as optogenetics1, electrophysiology2,3,4,5, plasticity6,7,8,9, neurotoxicity/neuroprotection10,11,12,13, cell therapy14, drug screening15,16,17, genetics and gene editing12,18,19,20, among others, as a strategy for better understanding neurological diseases during adulthood.
The comprehension of mechanisms underlying human brain pathologies depends on experimental strategies that require a large number of subjects. Conversely, in the case of slice cultures, although access to human samples is still difficult, the possibility of generating up to 50 slices from a single cortical sample partially circumvents the requirement of recruiting multiple volunteers by increasing the number of replicates and performed assays per collected tissue21.
Several protocols for brain organotypic/slice cultures have been described, ranging from the classical oculo drafts22,23 to roller tube24,25,26, semi-permeable membranes interface27,28,29,30, and free-floating slices31,32. Depending on the particularities of an experimental design, each technique has its own advantages and disadvantages. Short-term, free-floating slices cultures from adult human brains is in some cases advantageous over the method used by Stoppini et al.27, if considering the fact that although long-term cell survival in vitro is usually a major concern when evaluating a culture method, in many experiments only short periods of time in culture are needed12,31,32,33,34,35. Under these conditions, the use of free-floating cultures presents the advantage of being simpler and more cost-effective, as well as more accurately resembling the original human tissue condition than slices kept in culture over 2-3 weeks.
Despite the potential of slice cultures to neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly from human subjects. This article describes a protocol to use collected brain tissue from living human donors submitted to resective brain surgery to prepare free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are detailed. This protocol has been proven valuable for analyzing viability and neuronal function in investigations on the mechanisms of neuropathologies linked to adulthood.

<table border="0" cellpadding="0" cellspacing="0" style="width:1571px;" width="1570">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">2-Propanol</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">1096341000</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Acrylamide/Bis-Acrylamide, 30% solution</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">A3449&#160;</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Ammonium persulfate</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">A3678-25G</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Amphotericin B</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:344px;">15290-018</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Antibody anti-ERK 2 (rabbit)</td>
			<td>Santa Cruz Biotecnology</td>
			<td>sc-154</td>
			<td>Dilution 1:1,000 in BSA 2.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Antibody anti-pERK (mouse)</td>
			<td>Santa Cruz Biotecnology</td>
			<td>sc-7383</td>
			<td>Dilution 1:1,000 in BSA 2.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BDNF</td>
			<td>Sigma Aldrich</td>
			<td>SRP3014</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Bovine Serum Albumin</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">A7906</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Bradford 1x Dye Reagent</td>
			<td style="width:200px;">BioRad</td>
			<td style="width:344px;">500-0205</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">EDTA</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">T3924</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose</td>
			<td>Merck</td>
			<td>108337</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#39;s Balanced Salts</td>
			<td>Sigma Aldrich</td>
			<td>H1387-10X1L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Hydrochloric acid</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">1003171000</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Hydrogen Peroxide (H2O2)</td>
			<td style="width:200px;">Vetec</td>
			<td align="right" style="width:344px;">194</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Mouse IgG, HRP-linked whole Ab (anti-mouse)</td>
			<td style="width:200px;">GE</td>
			<td style="width:344px;">NA931-1ML</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">NaCl</td>
			<td style="width:200px;">Merck</td>
			<td align="right" style="width:344px;">1064041000</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal A</td>
			<td>Gibco</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Non-fat dry milk (Molico)</td>
			<td style="width:200px;">Nestl&#233;</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Used for membrane blocking</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">PBS Buffer pH 7,2</td>
			<td>Laborclin</td>
			<td align="right">590338</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicilin/Streptomicin</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:521px;">Potassium Chloride</td>
			<td>Merck</td>
			<td align="right">1049361000</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:521px;">Prime Western Blotting Detection Reagent</td>
			<td style="width:200px;">GE</td>
			<td style="width:344px;">RPN2232</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Rabbit IgG, HRP-linked whole Ab (anti-rabbit)</td>
			<td>GE</td>
			<td>NA934-1ML</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:521px;">SDS</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">L5750</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:521px;">TEMED</td>
			<td style="width:200px;">GE</td>
			<td style="width:344px;">17-1312-01</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Thiazolyl Blue Tetrazolium Bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>M5655</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:521px;">Tris</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">T-1378</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:521px;">Triton x-100</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:344px;">X100</td>
			<td style="width:505px;">Used in RIPA buffer</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:521px;">Ultrapure Water</td>
			<td style="width:200px;">Millipore</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Sterile water, derived from MiliQ water purification system</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:521px;">Equipment and Material</td>
			<td style="width:200px;"></td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:50px;">24-well plates</td>
			<td>Corning</td>
			<td>CL S3526</td>
			<td>Flat Bottom with Lid</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Amersham Potran Premium (nitrocellulose membrane)&#160;</td>
			<td>GE</td>
			<td>29047575</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:521px;">Carbogen Mixture</td>
			<td style="width:200px;">White Martins</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">95% O2, 5% CO2</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:46px;width:521px;">CO2 incubator</td>
			<td style="width:200px;">New Brunswick Scientific</td>
			<td style="width:344px;">CO-24</td>
			<td style="width:505px;">Incubation of slices 5% CO2, 36&#186;C</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:521px;">Microplate Reader</td>
			<td style="width:200px;">Molecular Devices</td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:521px;">Microtubes</td>
			<td style="width:200px;">Greiner</td>
			<td style="width:344px;">001608</td>
			<td style="width:505px;">1,5mL microtube</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:521px;">Motorized pestle</td>
			<td style="width:200px;">Kimble Chase</td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:521px;">Plastic spoon</td>
			<td style="width:200px;"></td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Size of a dessert spoon</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:521px;">Razor Blade</td>
			<td style="width:200px;">Bic</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Chrome Platinum, used in slicing with vibratome</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:37px;width:521px;">Scalpel Blade</td>
			<td style="width:200px;">Becton Dickinson (BD)</td>
			<td style="width:344px;">Number 24</td>
			<td style="width:505px;">Used for slicing of tissue; recommended same size or smaller</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:521px;">Superglue (Loctite Super Bonder)</td>
			<td style="width:200px;">Henkel</td>
			<td style="width:344px;"></td>
			<td style="width:505px;">Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6&#39;-di-terc-butil-2,2&#39;-metilenodi-p-cresol; homopolymer</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">Vibratome&#160;</td>
			<td>Leica</td>
			<td>14047235612 - VT1000S</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:37px;width:521px;">Name of Material/ Equipment for Immunohistochemistry</td>
			<td style="width:200px;"></td>
			<td style="width:344px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;">Antibody anti-NeuN (mouse)</td>
			<td>Millipore&#160;</td>
			<td>MAB377</td>
			<td>Dilution 1:1,000 in Phosphate Buffer</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:521px;">Antibody anti-GFAP (mouse)</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">MAB360</td>
			<td>Dilution 1:1,000 in Phosphate Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Antibody anti-Iba1 (rabbit)</td>
			<td style="width:200px;">Abcam</td>
			<td style="width:344px;">EPR16588 - ab178846</td>
			<td>Dilution 1:2,000 in Phosphate Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Biotinylated anti-mouse IgG Antibody (H+L)</td>
			<td style="width:200px;">Vector</td>
			<td style="width:344px;">BA-9200</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">DAB</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">D-9015</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Entellan</td>
			<td style="width:200px;">Merck</td>
			<td align="right" style="width:344px;">107960</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Ethanol</td>
			<td style="width:200px;">Merck</td>
			<td style="width:344px;">1.00983.1000</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Gelatin</td>
			<td style="width:200px;">Synth</td>
			<td style="width:344px;">00G1002.02.AE</td>
			<td style="width:505px;">Used for coating slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtome</td>
			<td>Leica</td>
			<td>SM2010R</td>
			<td>Equipped with Freezing Stage (BFS-10MP, Physiotemp), set to -40&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum</td>
			<td>Jackson Immuno Research</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Paraformaldehyde</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:344px;">158127</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Rabbit IgG, HRP-linked whole Ab (anti-rabbit)</td>
			<td>GE</td>
			<td>NA934-1ML</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slides (Star Frost)</td>
			<td>Knittel Glaser</td>
			<td></td>
			<td>Gelatin coated slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td style="width:200px;">Vetec</td>
			<td>60REAVET017050</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Vectastain ABC HRP Kit (Peroxidase, Standard)</td>
			<td style="width:200px;">Vector</td>
			<td style="width:344px;">PK-4000, Kit Standard</td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:521px;">Xylene</td>
			<td style="width:200px;">Synth</td>
			<td style="width:344px;">01X1001.01.BJ</td>
			<td style="width:505px;"></td>
		</tr>
	</tbody>
</table>

short-term free-floating slice cultures from the adult human brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A critical aspect to evaluate the quality and health of cultured slices is the presence and typical morphology of the expected neural cell types, neurons, and glial cells. The typical architecture of the human cortical lamination was observed in a slice at DIV4, revealed by neuronal immunolabeling (Figure 2D). In addition, the expected presence of microglia and astroglia (Figure 2B,C) was also observed. These results demonstrate...

Watch this Scientific Journal Video about Short-Term Free-Floating Slice Cultures from the Adult Human Brain at JoVE.com

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

short-term-free-floating-slice-cultures-from-the-adult-human-brain

Research

JoVE Journal

Neuroscience

8.7K Views.  University of São Paulo. A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Video: Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.