We would like to thank Dr Vasiliki Economopoulos for her in-house script to measure the fuluorescence signal in ImageJ. We would also want to thank the CITIUS (University of Seville) and all their personnel for their support during the demonstration. This work was supported by the Spanish FEDER I + D + i-USE, US-1264152 from University of Seville, and the Ministerio de Ciencia, Innovaci&#243;n y Universidades PID2021-126090OA-I00

The four different mushroom variants described in this protocol were obtained from a commercial source (see Table of Materials).
1. Isolation of fungal &#946;-glucans
<ol>
	<li>Extraction and isolation of soluble mushroom polysaccharides 
	NOTE: Soluble mushroom polysaccharides (SMPs) were obtained according to the procedure schematically shown in Figure 1.
	<ol>
		<li>Gently rinse fresh P. ostreatus, P. djamor, H. erinaceus,&#160;and G. lucidum fruiting bodies (about 2,000 g/mushroom) in distilled water five times.</li>
		<li>Dry the fruit bodies at 50 &#177; 2 &#176;C in a conventional air-drying oven until a constant weight is reached (~24 h).</li>
		<li>Ground the dried mushrooms in a blade mill, obtaining about 200 g of powder from each mushroom.</li>
		<li>Suspend 100 g of mushroom powder (MP) (P. ostreatus, P. djamor, H. erinaceus, and G. lucidum) in 1,000 mL of H2Od. Then, autoclave at 121 &#176;C for 15 min, and finally leave at room temperature for 30 min.</li>
		<li>Centrifuge the resulting suspension at 6,000 x g for 10 min at 4 &#176;C.</li>
		<li>Dry the precipitate containing insoluble mushroom polysaccharides (IMPs) at 50 &#177; 2 &#176;C in an air-drying oven for 24 h.</li>
		<li>Discard the precipitate and keep the supernatant. Concentrate the supernatant 10 times in a rotary evaporator.</li>
		<li>Precipitate the concentrate containing SMPs with ethanol (80% final concentration) at 4 &#176;C overnight.</li>
		<li>Centrifuge the ethanol suspension at 6,000 x g for 15 min at 4 &#176;C, retain the pellet (precipitate), and discard the supernatant with a pipette.</li>
		<li>Wash the precipitate three times with 80% ethanol before dissolving it in H2Od (10% w/v). Adjust the pH to 6.5/7 and the temperature to 37 &#176;C, and treat with 2 U and 4 U of &#945;-amylase and glucoamylase, respectively, to solubilize &#945;-glucans following the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>After the treatment with &#945;-amylase/glucoamylase, adjust the pH and temperature to 8.0 and 50 &#176;C, respectively, and treat with alcalase (2.5 U/g of protein) (see Table of Materials) to solubilize the proteins. 
		NOTE: This sequential enzymatic treatment removes most &#945;-glucans and proteins that co-precipitate with &#946;-glucans in the ethanol precipitation.</li>
		<li>After hydrolysis, centrifuge the hydrolysate at 6,000 x g for 15 min at 4 &#176;C, and the clean supernatant concentrate five times in a rotary evaporator. Precipitate again with 80% ethanol.</li>
		<li>Solubilize the resulting precipitate in H2Od and dialyze in distilled water for 24 h using cellulose tubing membranes (12,000 Da cut-off membranes; see Table of Materials) to remove low molecular weight molecules. Recover the water-soluble portion and freeze-dry it to produce soluble &#946;-glucans (S&#946;Gs).</li>
	</ol>
	</li>
	<li>Sugar and protein measurement
	<ol>
		<li>Measure the total sugar content of each fraction by the phenol-sulfuric acid method, using glucose as standard8. 
		NOTE: Quantitation of &#946;-glucan content may also be done by using the &#946;-glucan assay kit (mushroom and yeast; see Table of Materials), based on enzymatic hydrolysis and the activity of oxidoreductases: namely exo-1,3-&#946;-glucanase, glucose oxidase, &#946;-glucosidase, and peroxidase, with the subsequent formation of the quinoneimine. Follow the manufacturer&#39;s instructions, with slight modifications.</li>
		<li>Use 18 MH2SO4 instead of 12 MH2SO4.</li>
		<li>Evaluate the content of the total glucans and &#945;-glucans separately.</li>
		<li>Measure the resulting &#946;-glucan values as the difference between the total glucan and &#945;-glucan (triplicate) values following the Kjeldahl protocol. In certain cases, protein content can be determined by the Lowry method, using albumin to plot the calibration curve11,12.</li>
	</ol>
	</li>
	<li>Ultraviolet absorption spectroscopy analysis
	<ol>
		<li>Obtain the S&#946;G ultraviolet (UV) spectra using a UV-visible spectrophotometer (see Table of Materials) by scanning the samples in the 200-400 nm region (Figure 2).</li>
		<li>Prepare 1.0 mg/mL of each S&#946;G in H2Od, transfer the solution to a quartz cuvette, and scan at room temperature.</li>
	</ol>
	</li>
	<li>Molecular weight distribution analysis
	<ol>
		<li>Estimate the homoegeneity of S&#946;Gs and molecular weight of polymers by size exclusion chromatography (SEC) using a high-performance liquid chromatography (HPLC) system (see Table of Materials) equipped with a refractive index detector and an ultra-hydrogel linear gel-filtration column (300 mm x 7.8 mm; Figure 3).</li>
		<li>Perform the assay at 40 &#176;C using deionized water as eluent at a flow rate of 0.5 mL/min-1 and dextrans (110, 80, and 50 kDa) as standards (see Table of Materials). Collect a 5 mL fraction.</li>
	</ol>
	</li>
	<li>Fourier-transform infrared (FTIR) analysis
	<ol>
		<li>Record the infrared spectra (Figure 4) on an FTIR spectrometer in the range of 4000-500 cm-1. The samples should be previously mixed with KBr to form films (standard FTIR procedure; see manufacturer&#39;s instructions and Table of Materials).</li>
	</ol>
	</li>
	<li>Molecular composition analysis
	<ol>
		<li>Estimate the molecular compositions of S&#946;Gs by high-performance thin-layer chromatography (HPTLC) as well as gas chromatography coupled to mass spectrometry (GC-MS), following standard procedures12.</li>
	</ol>
	</li>
</ol>
2. In vitro study of &#946;-glucan-induced microglia stimulation
<ol>
	<li>Cell culture of mouse glioblastoma and microglia cells in 8-well chamber slides 
	NOTE: This protocol is specific for GL261 (glioblastoma) and BV2 (microglia) cell lines. However, with slight modifications, these steps could potentially be used to study other cancer and immune cell lines.
	<ol>
		<li>Prepare Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) complete medium modified with L-glutamin, 4.5 g/L D-glucose, and without pyruvate. Add 10% of fetal bovine serum (FBS) and 1% penicillin/streptomycin (see Table of Materials). Pre-warm the material in a water bath at 37 &#176;C for 15 min.</li>
		<li>Thaw frozen BV2 and GL261 aliquots into a water bath (37 &#176;C) for 2 min, and just before they completely thaw, carry them into a laminar flow hood and plate the cells into two different sterile T25 flasks (one for each cell line).</li>
		<li>Incubate the T25 flasks at 37 &#176;C, 5% CO2 until the culture is confluent. 
		NOTE: Depending on the freezing conditions and the time under cryopreservation, the time until confluence may vary. These cell lines usually require between 3 to 5 days to reach confluency in a T75 flask.</li>
		<li>After the BV2 cell culture becomes confluent, transfer it into 8-well chamber slides 0.6 x 106 cells/well. Keep the 8-well chamber slides in the incubator for 24 h.</li>
		<li>Once the microglia cells are plated into the 8-well chamber slides, repeat the same protocol with the GL261 cells.</li>
	</ol>
	</li>
	<li>Activation of microglia with &#946;-glucans
	<ol>
		<li>Coat the BV2 cells with four different &#946;-glucans (P. ostreatus, P. djamor, G. lucidum, and H. erinaceus) at a 0.2 mg/mL concentration for 72 h. One experimental condition must remain untreated (normal medium), acting as the control group.</li>
		<li>Collect the supernatant with a pipette after 72 h and pass the remaining volume through a 0.20 &#181;m syringe filter. Then, freeze the supernatant at -80 &#176;C for at least 24 h.</li>
	</ol>
	</li>
	<li>Treatment of GL261 with pre-activated microglia-conditioned medium
	<ol>
		<li>Once the GL261 is 80% confluent within the 8-well chamber slides, add &#946;-glucan-treated microglial medium (step 2.2.2) at a final volume concentration of 25% for 72 h (total volume: 250 &#181;l/well).</li>
		<li>Remove the medium after 72 h incubation and discard it.</li>
		<li>Wash the cells with phosphate buffer saline (PBS; pH 7.4, 0.1 M) three times for 5 minutes.</li>
		<li>Fix the cells by adding 200 &#181;L of 4% paraformaldehyde (PFA) at 4 &#176;C for 15 min. 
		NOTE: Depending on the different antibodies that might be used for immunofluorescence, fixation methods may differ from typical 4% PFA. Alcohol-based fixatives may be more efficient in preserving certain epitopes.</li>
	</ol>
	</li>
	<li>Immunofluorescence study
	<ol>
		<li>Wash the samples with PBS with triton X (PBST; 0.01%) for 10 min three times.</li>
		<li>Remove the PBST and add bovine serum albumin (BSA) blocking buffer 10% in PBST (Table 1) for 30 min at room temperature.</li>
		<li>Remove the blocking buffer and add 200 &#181;L per well of PBS containing the primary antibodies mixture (1:500 rat Ki-67 monoclonal antibody and 1:500 rabbit cleaved caspase-3 antibody; see Table of Materials). Incubate overnight at 4 &#176;C.</li>
		<li>After 24 h of incubation at 4 &#176;C, leave the samples at room temperature for 30 min.</li>
		<li>Wash the wells three times for 10 min with PBS on a shaker (low speed).</li>
		<li>Remove the PBS and replace with 200 &#181;L per well of PBS containing the mixture of secondary antibodies (1:200 donkey anti-rat IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 and 1:200 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647; see Table of Materials) for 45 min at room temperature in the dark.</li>
		<li>Wash the samples with PBS for 10 min on a multipurpose shaker.</li>
		<li>Remove the PBS and add 200 &#181;L per well of 4&#8242;,6-diamidino-2-phenylindole (DAPI) diluted in PBS (1:5,000) for 1 min.</li>
		<li>Remove DAPI (see Table of materials) and wash the cells for 5 min in PBS.</li>
		<li>Remove the well frame and add 50 &#181;L of PBS:glycerol (1:1) on each well and cover with a coverslip.</li>
		<li>Seal the slides with nail polish.</li>
		<li>Acquire images at 20x using a confocal microscope system (see Table of materials).</li>
	</ol>
	</li>
</ol>
3. Quantification and analysis of tumor cell proliferation and apoptosis
NOTE: In order to measure the potential effect of the different &#946;-glucans on tumor cell proliferation and apoptosis, an in-house script was used in ImageJ software to quantify the number of positive pixels of Ki67 (proliferation) and cCasp3 (apoptosis)13.
<ol>
	<li>Open ImageJ. Click on the Plugins button. Click on Coloc2, a plugin previously installed in the plugin folder, and finally select the image to analyze11. 
	NOTE: This plugin was available following previous contact with Dr. Vasiliki Economopoulos (veconom@uwo.ca). Instead of the script, both ImageJ and Fiji software have different tools for colocalization analysis (see Table of materials), with similar properties.</li>
	<li>Set thresholds according to the control (untreated, DMEM only) conditions. Click on the OK button. 
	NOTE: In order to avoid background and intensity discrepancies, all images must be taken under the same conditions. Preferably, imaging sessions should be performed on the same day, and microscope parameters unaltered across images.</li>
	<li>Ensure that the resulting images of the colocalized pixels and a summary window providing the percentage or raw number of pixels above the threshold pop up. Normalize the results with respect to the control (untreated) conditions. 
	NOTE: All data are given as mean &#177; SEM. Statistical analysis was performed using graphing and analysis software (see Table of Materials). A one-way ANOVA with Tukey&#39;s multiple comparison test was used. Errors are represented as standard error of the mean (s.e.m.) (*p &#60; 0.05, **p &#60; 0.01).</li>
</ol>

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There are no competing interests to declare.

This work describes the use of well-established techniques to successfully isolate, purify, and characterize the content of S&#946;Gs from four different fungi. The results showed how after hot water extraction of SMPs, obtained from P. ostreatus, P. djamor, G. lucidum, and H. erinaceus,&#160;followed by hydrolytic treatment with &#945;-amylase, glucosidase, and protease, the content of &#945;-glucan and protein were reduced, thus significantly enriching the amount of pure S&#946;Gs.</...

Despite the advent of novel achievements in the field of neuro-oncology, the life expectancy of glioblastoma patients remains meager. Gold-standard therapies against brain tumors are based on the amalgamation of surgery, radiotherapy, and chemotherapy. However, in the last decade, immunotherapy has emerged as a powerful strategy to treat different types of cancer1. Thus, the possibility of harnessing the body&#39;s immune response against tumor cells has recently become the fourth pillar of oncology.
It has long been known that one of the biggest challenges in the field is to overcome the strong immunosuppression found within the tumor microenvironment2. Particularly, in the case of glioblastoma, one of the most common and aggressive forms of brain cancer, unraveling key pathways that orchestrate such pro-tumoral scenarios and finding novel compounds that could counteract the depressing response of the immune system might pave the way for future therapies against this incurable disease.
The brain possesses its own immune system cells, and the most relevant cell type are microglia. These cells have been proven to have a rather complex behavior across different central diseases3. In the case of primary brain tumors (e.g., glioblastoma), these cells are shifted toward an anti-inflammatory phenotype that supports tumor cells to colonize the brain parenchyma3. Numerous publications have enhanced the major role of these cells during tumor progression. One of the main reasons for this is that glioma-associated microglia and infiltrated macrophages (GAMs) account for one-third of the total tumor mass, thus suggesting the unequivocal influence of their activation states during brain tumor progression4,5.
In that vein, fungal &#946;-glucans have been described as potent molecules triggering effective immune responses, including phagocytosis and pro-inflammatory factors production, leading to the elimination of pernicious agents6,7,8,9,10. Fungal &#946;-glucans have generally been studied using extracts from different mushroom parts. However, the attribution of specific effects requires its purification to avoid ambiguities and to be able to understand the mechanism of action of such molecules as immunomodulatory agents8.
In this work, soluble &#946;-glucans are purified from the fruiting body of four different mushrooms, regularly employed as edible (Pleurotus ostreatus and Pleurotus djamor) and as medicinal (Ganoderma lucidum and Hericium erinaceus) mushrooms. In particular, these four mushrooms have great use in the food and pharmaceutical industry and were produced within an environmentally friendly circular economy in a commercial enterprise (see Table of Materials).
In order to lay the foundation for the future use of fungal &#946;-glucans in brain cancer therapies, well-defined purification strategies and preclinical studies delving into their putative interaction with immune system cells are essential to evaluate their potential role as anti-tumor mediators. This work describes the numerous steps of isolation and purification needed to retrieve the soluble &#946;-glucans contained within the fruiting bodies of the selected mushroom. Once successfully purified, microglia cells are activated to enhance their inflammatory phenotype. Mouse glioblastoma cells (GL261) are coated with a different microglia-conditioned medium, previously treated with these extracts, and then its effect on tumor cells&#39; behavior is evaluated. Interestingly, pilot studies from our lab (data not shown) have uncovered how pro-inflammatory microglia may slow tumor cell migration and invasion properties not only in glioblastoma cells but also in other cancer cell lines. This multidisciplinary work may provide a useful tool for oncology researchers to test promising compounds able to boost the immune response in many different types of tumors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well chamber slides</td>
			<td>Thermo Fisher, USA</td>
			<td>171080</td>
			<td></td>
		</tr>
		<tr>
			<td>Air-drying oven</td>
			<td>J.P. Selecta S.A., Spain</td>
			<td>2000210</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich, St. Louis</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcalase</td>
			<td>Novozymes, Denmark</td>
			<td>protease</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Thermofisher, USA</td>
			<td>A32731</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647</td>
			<td>Thermofisher, USA</td>
			<td>A32728</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade mill</td>
			<td>Retsch, Germany</td>
			<td>&#160;SM100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MERK, Germany</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose tubing membrane</td>
			<td>Sigma-Aldrich, St. Louis</td>
			<td>D9402</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>MERK, Germany</td>
			<td>Eppendorf, 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Colocalisation pluggins</td>
			<td>ImageJ</td>
			<td></td>
			<td>(https://imagej.net/imaging/colocalization-analysis&#160;)</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MERK, Germany</td>
			<td>28718-90-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrans</td>
			<td>Pharmacosmos, Holbalk, Denmark</td>
			<td>Dextran 410, 80, 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s modified Eagle&#180;s medium, Gluta MAXTM</td>
			<td>Gibco, Life Technologies, Carlsbad, CA, USA</td>
			<td>10564011</td>
			<td></td>
		</tr>
		<tr>
			<td>Extenda (&#945;- Amylase/Glucoamylase)</td>
			<td>Novozymes, Denmark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco, Life Technologies, Carlsbad, CA, USA</td>
			<td>A4736301</td>
			<td></td>
		</tr>
		<tr>
			<td>FT-IR spectromete</td>
			<td>Bruker-Vertex, Switzerland</td>
			<td>VERTEX 70v</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphing and analysis software</td>
			<td>GraphPad Prism (GraphPad Software, Inc.)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Waters Corp, Milford, MA, USA</td>
			<td>Waters 2695 HPLC</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Eppedorf</td>
			<td>Galaxy 170S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectometer</td>
			<td>Q Exactive GC, Thermo Scientific</td>
			<td>725500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MERK, Germany</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich, St. Louis</td>
			<td>P4458</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Crison, Barcelona, Spain</td>
			<td>Basic 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Gibco, Life Technologies, Carlsbad, CA, USA</td>
			<td>1010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Cleaved Caspase-3 (Asp175) Antibody</td>
			<td>Abcam, UK</td>
			<td>ab243998</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Ki-67 Monoclonal</td>
			<td>Thermofisher, USA</td>
			<td>MA5-14520</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>B&#252;chi Ib&#233;rica S.L.U., Spain</td>
			<td>El Rotavapor R-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-hydrogel linear gel-filtration column (300 mm x 7.8 mm)</td>
			<td>Waters Corp, Milford, MA, USA</td>
			<td>WAT011545</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Visible spectrophotometer</td>
			<td>Amersham Bioscience, UK</td>
			<td>Ultrospec 2100 pro</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaMount</td>
			<td>Vector Laboratories, C.A, USA</td>
			<td>H-5000-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>J.P. Selecta S.A., Spain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 7 DUO Confocal Microscope System.</td>
			<td>Zeiss, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glucan Assay Kit</td>
			<td>Megazyme, Bray, Co. Wicklow, Ireland</td>
			<td>K-BGLU</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glucans</td>
			<td>Setas y Hongos del Sur, S.L.</td>
			<td></td>
			<td>Supplied the four variants of mushrooms</td>
		</tr>
	</tbody>
</table>

isolation and purification of fungal &#946;-glucan as an immunotherapy strategy for glioblastoma

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor microenvironment. Immunotherapy has emerged as an effective strategy to turn the immune system response against tumor cells. Glioma-associated macrophages and microglia (GAMs) are major drivers of such anti-inflammatory scenarios. Therefore, enhancing the anti-cancerous response in GAMs may represent a potential co-adjuvant therapy to treat glioblastoma patients. In that vein, fungal &#946;-glucan molecules have long been known as potent immune modulators. Their ability to stimulate the innate immune activity and improve treatment response has been described. Those modulating features are partly attributed to their ability to bind to pattern recognition receptors, which, interestingly, are greatly expressed in GAMs. Thus, this work is focused on the isolation, purification, and subsequent use of fungal &#946;-glucans to enhance the tumoricidal response of microglia against glioblastoma cells. The mouse glioblastoma (GL261) and microglia (BV-2) cell lines are used to test the immunomodulatory properties of four different fungal &#946;-glucans extracted from mushrooms heavily used in the current biopharmaceutical industry: Pleurotus ostreatus, Pleurotus djamor, Hericium erinaceus,&#160;and Ganoderma lucidum. To test these compounds, co-stimulation assays were performed to measure the effect of a pre-activated microglia-conditioned medium on the proliferation and apoptosis activation in glioblastoma cells.

Successful purification of &#946;-glucans 
The mass of MP, SMPs, and S&#946;Gs obtained from fruiting bodies of P. ostreatus, P. djamor, G. lucidum, and H. erinaceus following the extraction and purification process is summarised in Table 1. The basic composition (total carbohydrates, &#946;-glucans, and protein) of MP, SMPs, and S&#946;Gs obtained from the fungi is depicted in Table 2. These results show how the protocol allowed ...

Watch this Scientific Journal Video about Isolation and Purification of Fungal ÃŽÂ²-Glucan as an Immunotherapy Strategy for Glioblastoma at JoVE.com

Isolation and Purification of Fungal &#946;-Glucan as an Immunotherapy Strategy for Glioblastoma

The present protocol describes the purification steps and subsequent studies of four different fungal &#946;-glucans as potential immunomodulatory molecules that enhance the anti-tumoral properties of microglia against glioblastoma cells.

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor ...

isolation-purification-fungal-glucan-as-an-immunotherapy-strategy-for

Research

JoVE Journal

Neuroscience

1.7K Views.  Universidad de Sevilla. The present protocol describes the purification steps and subsequent studies of four different fungal β-glucans as potential immunomodulatory molecules that enhance the anti-tumoral properties of microglia against glioblastoma cells.

Video: Isolation and Purification of Fungal β-Glucan as an Immunotherapy Strategy for Glioblastoma

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Using Chick Embryo Brain as a Model to Study Human Glioblastoma Cell Behavior

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. 
Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 &#181;m) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 &#181;m) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. 
Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...