This work was funded by the US Department of Veterans Affairs BLR&amp;D IK2 BX001686 (to TAB), and grants from the University of Minnesota Healthy Foods, Healthy Lives Institute (to CMD, JPN, and TAB) and the Minnesota Veterans Medical Research &amp; Education Foundation (to TAB). We thank Drs. Philippe Marambaud (Feinstein Institute for Medical Research, Manhasset, NY) and Weihua Zhao (Methodist Hospital, Houston, TX) for providing the BV2 cell line.

1. Microglial Cell Culture Maintenance
<ol>
	<li>Warm cell culture medium (Dulbecco&#39;s Modified Eagle Medium; DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/neomycin (PSN) to 37 &#176;C.</li>
	<li>Obtain frozen stock of BV2 microglial cells at passage 18 - 25 from storage at -80 &#176;C. Rapidly thaw cells in 37 &#176;C water bath.</li>
	<li>Gently transfer cells to a 75 cm2 vented flask containing 10 ml cell culture media.</li>
	<li>Incubate flask in 5% CO2 at 37 &#176;C. Aspirate media after 24 hr and replace with fresh media. Grow cells until approximately 70 - 80% confluency.</li>
</ol>
2. Counting and Plating Cells
<ol>
	<li>In a 37 &#176;C water bath, warm DMEM and 1x-trypsin-EDTA solution.</li>
	<li>Aspirate media from cells and add 500 &#181;l of trypsin. Incubate at 37&#176;C for 2 - 5 min.</li>
	<li>Using a scraper, remove cells from the flask and suspend in 5 ml of DMEM. Pass cells through a 70 &#181;m strainer into a 50 ml tube three times.</li>
	<li>Count cells using a hemocytometer and seed 8,000 cells/well in a black walled clear bottom plate in a final volume of 200 &#181;l DMEM supplemented with 10% FBS and 1% PSN. Incubate plate in 5% CO2 at 37 &#176;C for 24 hr.</li>
	<li>Remove old DMEM and replace with 200 &#181;l of warmed DMEM supplemented with 1% PSN to serum starve microglia. Incubate plate in 5% CO2 at 37 &#176;C for 24 hr.</li>
</ol>
3. Activating Microglia
<ol>
	<li>In separate tubes, dilute AgNPs (0.01, 0.05 or 0.1 &#181;g/ml) and vehicle/neutral control (sodium citrate, 0.04 mM) in serum-free DMEM supplemented with 1% PSN to final working concentrations.</li>
	<li>Remove 100 &#181;l of media from each well and replace with 100 &#181;l of appropriate treatment compound.</li>
	<li>Incubate plate in 5 % CO2 at 37 &#176;C for 24 hr.</li>
</ol>
4. Filtering Conditioned Media
<ol>
	<li>Collect 200 &#181;l of media supernatant from each well and transfer into a filter (molecular weight cutoff of 10 kDa) with collection tube (1.5 to 2 ml capacity).</li>
	<li>Centrifuge at 14,000 x g at room temperature for 15 min.</li>
	<li>Discard the flow-through (supernatant with particles) and place the filter upside-down into a new collection tube.</li>
	<li>Centrifuge at 1,000 x g at room temperature for 2 min. 
	NOTE: The resulting filtered media (containing secreted cytokines) is concentrated by six-fold.</li>
	<li>Bring the volume of the concentrate to 400 &#181;l with fresh DMEM supplemented with 10% FBS and 1% PSN and store on ice. 
	NOTE: To ensure residual AgNPs are removed from filtered media, generate an AgNP concentration based on the characteristic peak at about 390 - 420 nm 10. Briefly, prepare aliquots of unfiltered AgNPs (0, 0.01, 0.025, 0.05, 0.1, and 0.2 &#181;g/ml as described in step 3.1) and filtered vehicle control (sodium citrate, 0.04 mM) in serum-free DMEM. Aliquot 50 &#181;l of filtered samples and standards into a black walled clear bottom plate. Using a spectrophotometer, measure absorbance at 390 - 410 nm and compare readings to the AgNP concentration curve. If filtered samples have the same absorbance value as the vehicle control sample, it can be assumed residual AgNPs are removed.</li>
	<li>Use half of the filtered media to determine TNF-&#945; secretion following instructions from a commercially available kit.</li>
</ol>
5. Hypothalamic Cell Culture Maintenance
<ol>
	<li>Warm DMEM cell culture medium supplemented with 10% FBS and 1% PSN to 37 &#176;C.</li>
	<li>Obtain frozen stock of hypothalamic (mHypo-A1/2) cells at passage 18 - 25 stored at 80 &#176;C. Rapidly thaw cells in 37 &#176;C water bath.</li>
	<li>Gently transfer cells to a 75 cm2 vented flask containing 10 ml cell culture media.</li>
	<li>Incubate flask in 5% CO2 at 37 &#176;C. Aspirate media after 24 hr and replace with fresh media. Grow cells until approximately at 70 - 80% confluency.</li>
</ol>
6. Determining Hypothalamic Cell Toxicity
<ol>
	<li>Warm DMEM and 1x-trypsin-EDTA solution in a 37 &#176;C water bath.</li>
	<li>Aspirate media from hypothalamic cells and add 500 &#181;l of trypsin. Incubate at 37 &#176;C for 2 - 5 min. Remove cells from flask using a scraper as described above in step 2.</li>
	<li>Count cells using a hemocytometer and plate hypothalamic cells at 5,000 cells/well in a black walled clear bottom plate in a final volume of 200 &#181;l DMEM supplemented with 10% FBS and 1% PSN and incubate overnight in 5% CO2 at 37 &#176;C.</li>
	<li>Remove 100 &#181;l of old media using a multichannel pipetter and add 100 &#181;l of filtered concentrated conditioned media, to a final concentration of 1x.</li>
	<li>Incubate plate in 5% CO2 at 37 &#176;C for 24 hr.</li>
	<li>Add 22 &#181;l of resazurin reagent and incubate plate for 20 min in 5% CO2 at 37 &#176;C.</li>
	<li>Using a multimode spectrophotometer, record fluorescence (560 nm EX/590 nm EM) to measure cell viability. Report results as relative fluorescence units (RFU).</li>
</ol>

<ol><li>Block, M. L., Calderon-Garciduenas, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Air+pollution%3A+mechanisms+of+neuroinflammation+and+CNS+disease%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Air pollution: mechanisms of neuroinflammation and CNS disease.</a> Trends Neurosci. 32, 506-516 (2009).</li>
<li>Brochu, P., Bouchard, M., Haddad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+daily+inhalation+rates+for+health+risk+assessment+in+overweight%2Fobese+children%2C+adults%2C+and+elderly%5BTitle%5D)+AND+%22Risk+Anal%22%5BJournal%5D)">Physiological daily inhalation rates for health risk assessment in overweight/obese children, adults, and elderly.</a> Risk Anal. 34, 567-582 (2014).</li>
<li>Jerrett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traffic-related+air+pollution+and+obesity+formation+in+children%3A+a+longitudinal%2C+multilevel+analysis%5BTitle%5D)+AND+%22Environ+Health%22%5BJournal%5D)">Traffic-related air pollution and obesity formation in children: a longitudinal, multilevel analysis.</a> Environ Health. 13, 49(2014).</li>
<li>Kraft, A. D., Harry, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Features+of+microglia+and+neuroinflammation+relevant+to+environmental+exposure+and+neurotoxicity%5BTitle%5D)+AND+%22Int+J+Environ+Res+Public+Health%22%5BJournal%5D)">Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity.</a> Int J Environ Res Public Health. 8, 2980-3018 (2011).</li>
<li>Duffy, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+orexin+A+signaling+in+dietary+palmitic+acid-activated+microglial+cells%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">Role of orexin A signaling in dietary palmitic acid-activated microglial cells.</a> Neurosci Lett. 606, 140-144 (2015).</li>
<li>Butterick, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+a+caspase+multiplexing+assay+to+determine+apoptosis+in+a+hypothalamic+cell+model%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model.</a> J Vis Exp. , (2014).</li>
<li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+of+cell+viability+and+proliferation+in+hydrogel-encapsulated+system+by+resazurin+assay%5BTitle%5D)+AND+%22Appl+Biochem+Biotechnol%22%5BJournal%5D)">Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.</a> Appl Biochem Biotechnol. 162, 1996-2007 (2010).</li>
<li>Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., Bistoni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immortalization+of+murine+microglial+cells+by+a+v-raf%2Fv-myc+carrying+retrovirus%5BTitle%5D)+AND+%22J+Neuroimmunol%22%5BJournal%5D)">Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus.</a> J Neuroimmunol. 27, 229-237 (1990).</li>
<li>Belsham, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ciliary+neurotrophic+factor+recruitment+of+glucagon-like+peptide-1+mediates+neurogenesis%2C+allowing+immortalization+of+adult+murine+hypothalamic+neurons%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons.</a> FASEB J. 23, 4256-4265 (2009).</li>
<li>Paramelle, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+method+to+estimate+the+concentration+of+citrate+capped+silver+nanoparticles+from+UV-visible+light+spectra%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">A rapid method to estimate the concentration of citrate capped silver nanoparticles from UV-visible light spectra.</a> Analyst. 139, 4855-4861 (2014).</li>
<li>Wei, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+exposure+to+air+pollution+particles+increases+the+risk+of+obesity+and+metabolic+syndrome%3A+findings+from+a+natural+experiment+in+Beijing%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Chronic exposure to air pollution particles increases the risk of obesity and metabolic syndrome: findings from a natural experiment in Beijing.</a> FASEB J. , (2016).</li>
<li>Levesque, S., Surace, M. J., McDonald, J., Block, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Air+pollution+%26+the+brain%3A+Subchronic+diesel+exhaust+exposure+causes+neuroinflammation+and+elevates+early+markers+of+neurodegenerative+disease%5BTitle%5D)+AND+%22J+Neuroinflammation%22%5BJournal%5D)">Air pollution & the brain: Subchronic diesel exhaust exposure causes neuroinflammation and elevates early markers of neurodegenerative disease.</a> J Neuroinflammation. 8, 105(2011).</li>
<li>Block, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+outdoor+air+pollution+and+brain+health+workshop%5BTitle%5D)+AND+%22Neurotoxicology%22%5BJournal%5D)">The outdoor air pollution and brain health workshop.</a> Neurotoxicology. 33, 972-984 (2012).</li>
<li>Carson, M. J., Crane, J., Xie, A. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+CNS+microglia%3A+the+quest+to+identify+predictive+models%5BTitle%5D)+AND+%22Drug+Discov+Today+Dis+Models%22%5BJournal%5D)">Modeling CNS microglia: the quest to identify predictive models.</a> Drug Discov Today Dis Models. 5, 19-25 (2008).</li>
<li>Valdearcos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+dictate+the+impact+of+saturated+fat+consumption+on+hypothalamic+inflammation+and+neuronal+function%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Microglia dictate the impact of saturated fat consumption on hypothalamic inflammation and neuronal function.</a> Cell Rep. 9, 2124-2138 (2014).</li>
<li>Perry, V. H., Holmes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+priming+in+neurodegenerative+disease%5BTitle%5D)+AND+%22Nat+Rev+Neurol%22%5BJournal%5D)">Microglial priming in neurodegenerative disease.</a> Nat Rev Neurol. 10, 217-224 (2014).</li>
<li>Block, M. L., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+microglial+activation+and+progressive+dopaminergic+neurotoxicity%5BTitle%5D)+AND+%22Biochem+Soc+Trans%22%5BJournal%5D)">Chronic microglial activation and progressive dopaminergic neurotoxicity.</a> Biochem Soc Trans. 35, 1127-1132 (2007).</li>
<li>Vincenti, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+Microglia+Response+during+the+Time+Course+of+Chronic+Neurodegeneration%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Defining the Microglia Response during the Time Course of Chronic Neurodegeneration.</a> J Virol. 90, 3003-3017 (2015).</li>
<li>Grabert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+brain+region-dependent+diversity+and+selective+regional+sensitivities+to+aging%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Microglial brain region-dependent diversity and selective regional sensitivities to aging.</a> Nat Neurosci. 19, 504-516 (2016).</li>
<li>Lull, M. E., Block, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+activation+and+chronic+neurodegeneration%5BTitle%5D)+AND+%22Neurotherapeutics%22%5BJournal%5D)">Microglial activation and chronic neurodegeneration.</a> Neurotherapeutics. 7, 354-365 (2010).</li>
<li>Oeckinghaus, A., Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crosstalk+in+NF-kappaB+signaling+pathways%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Crosstalk in NF-kappaB signaling pathways.</a> Nat Immunol. 12, 695-708 (2011).</li>
<li>Gifford, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thiol-modified+gold+nanoparticles+for+the+inhibition+of+Mycobacterium+smegmatis%5BTitle%5D)+AND+%22Chem+Commun+%28Camb%29%22%5BJournal%5D)">Thiol-modified gold nanoparticles for the inhibition of Mycobacterium smegmatis.</a> Chem Commun (Camb). 50, 15860-15863 (2014).</li>
<li>Colella, M., Lobasso, S., Babudri, F., Corcelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Palmitic+acid+is+associated+with+halorhodopsin+as+a+free+fatty+acid.+Radiolabeling+of+halorhodopsin+with+3H-palmitic+acid+and+chemical+analysis+of+the+reaction+products+of+purified+halorhodopsin+with+thiols+and+NaBH4%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Palmitic acid is associated with halorhodopsin as a free fatty acid. Radiolabeling of halorhodopsin with 3H-palmitic acid and chemical analysis of the reaction products of purified halorhodopsin with thiols and NaBH4.</a> Biochim Biophys Acta. 1370, 273-279 (1998).</li>
<li>Sherry, B., Jue, D. M., Zentella, A., Cerami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+high+molecular+weight+glycosylated+forms+of+murine+tumor+necrosis+factor%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Characterization of high molecular weight glycosylated forms of murine tumor necrosis factor.</a> Biochem Biophys Res Commun. 173, 1072-1078 (1990).</li>
<li>PubChem Compound Database. , National Center for Biotechnology Information. 
 <a target="_blank" href="https://pubchem.ncbi.nlm.nih.gov/compound/104755">https://pubchem.ncbi.nlm.nih.gov/compound/104755</a> (2004).</li>
<li>Koenigsknecht-Talboo, J., Landreth, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+phagocytosis+induced+by+fibrillar+beta-amyloid+and+IgGs+are+differentially+regulated+by+proinflammatory+cytokines%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines.</a> J Neurosci. 25, 8240-8249 (2005).</li>
<li>McCarthy, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+novel+adult+murine+immortalized+microglial+cell+line+and+its+activation+by+amyloid-beta%5BTitle%5D)+AND+%22J+Neuroinflammation%22%5BJournal%5D)">Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta.</a> J Neuroinflammation. 13, (2016).</li>
<li>Schauer, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Source+apportionment+of+airborne+particulate+matter+using+organic+compounds+as+tracers%5BTitle%5D)+AND+%22Atmos+Environ%22%5BJournal%5D)">Source apportionment of airborne particulate matter using organic compounds as tracers.</a> Atmos Environ. 30, 3837-3855 (1996).</li>
<li>Kleeman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Source+apportionment+of+fine+%28PM1.8%29+and+ultrafine+%28PM0.1%29+airborne+particulate+matter+during+a+severe+winter+pollution+episode%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">Source apportionment of fine (PM1.8) and ultrafine (PM0.1) airborne particulate matter during a severe winter pollution episode.</a> Environ Sci Technol. 43, 272-279 (2009).</li>
<li>Borm, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+potential+risks+of+nanomaterials%3A+a+review+carried+out+for+ECETOC%5BTitle%5D)+AND+%22Part+Fibre+Toxicol%22%5BJournal%5D)">The potential risks of nanomaterials: a review carried out for ECETOC.</a> Part Fibre Toxicol. 3, (2006).</li>
</ol>

The authors have no conflicts to disclose.

Recent studies support that environmental exposure contributes to obesity and other neurodegenerative diseases 11,12. However, techniques used in previous studies are time consuming and expensive. Economic considerations, physiologically relevant delivery systems, ethical issues with extensive use of in vivo animal models, and difficulty translating findings into meaningful health advisories are a few of the major challenges that have impeded advancements in studying NP-induced neurotoxicity 13. Therefore, use of an in vitro microglial surrogate biosensor has become an essential tool for environmental exposure to neurotoxins 14. In the methods described above, we provide a direct way to determine how nanoparticles such as AgNPs activate microglia and induce neurotoxicity. It should be noted that because the cells are not fixed or lysed, other downstream applications including gene expression and Western blots could be performed on the microglia or neurons. In addition to performing a TNF-&#945; ELISA, the supernatant can be used to analyze an array of cytokines and secreted factors from the stimulated microglia.
Microglial activation can be a result of many different factors including environmental exposures, infection, ischemia, and injury resulting in morphological and physiological changes. Chronic M1 microglial activation is associated with neurodegenerative diseases such as obesity, Alzheimer's disease, and Parkinson disease 5,15-19. Further, the secretion of pro-inflammatory cytokines from microglia, including TNF-&#945;, contributes to the onset of surrounding neuronal cell death 5,15,20. Other markers that can be investigated after microglial activation are changes in reactive oxygen species (ROS) and nitrogen species 17. Because pro-inflammatory cytokines such as TNF-&#945; are regulated by the transcription factor nuclear factor kappa B (NF&#954;B) 21, other downstream targets within this pathway can be profiled using this protocol. Additionally, microglial activation (acute or chronic) is stimuli dependent. Therefore, the timing and stimuli should be taken into consideration when performing the assays. The main limitation to this assay is that it is an in vitro study. As such, results should be cautiously extrapolated, and overinterpretation of results avoided, until biological significance has been verified in vivo. However, utilizing an in vitro test such as the microglial model described here, as a pre-screening method is a crucial first step to understanding toxicity of nanoparticles. Additionally, the specific particle used in testing should be considered carefully when interpreting results. Our protocol utilizes manufactured single metal AgNPs to serve as a surrogate for environmental pollutant generated nanoparticles. These particles were chosen not to mirror real-world environmental pollutants, but as standardized control particles to validate the methodology. Environmental pollutants represent multiple species of NPs, therefore particle morphology (size, shape, charge) and type of NP (silver, gold, titanium, carbon) should be important aspects of experimental design. Without taking such considerations into account, results may not be physiologically relevant, and thus difficult to interpret.
Critical steps of this protocol are performing proper aseptic technique, using appropriate doses of stimuli, and using the correct filtration device. Considerations for troubleshooting when planning studies to evaluate microglial activation and neuronal cell death include determining appropriate concentrations and incubation time of the stimuli, cell densities, and incubation time for reagents used in the assay. To optimize this assay dose response and time course studies should be performed. Additionally, it is essential to choose the appropriate supernatant filtration device to ensure separation of nanoparticles from secreted cytokines. The filters used in this protocol are able to separate nanoparticles and proteins based on size 22,23. Since AgNPs have a molecular weight of 0.107 kDa and the pro-inflammatory cytokine TNF-&#945; is 17 kDa 24,25, using a filter that has a 10 kDa molecular weight cutoff therefore allows factors or proteins less than 10 kDa (including AgNPs) to be excluded from the conditioned media. To validate that AgNPs were completely removed from the conditioned media, absorbance measurements of the media were taken after filtration and compared to an AgNP standard curve. We can therefore conclude that the cytotoxic effects of the conditioned media was due to cytokines/proteins such as TNF-&#945; released from activated microglia, and not due to residual AgNPs 5.
The microglial model described here can be used to provide valuable insight into the role of microglia-induced cell death after nanoparticle exposure, and validates the effectiveness of utilizing microglia as a surrogate biosensor in determining nanoparticle toxicity 5. Modifications of this protocol can be used to fit other models of microglial-induced cell death including models of neurodegenerative disease, utilization of other cell types (primary, transfected, immortalized), or various alternate stimuli (fatty acids, amyloid-beta) 5,26,27. Although literature supports that BV2 microglia are well characterized and commonly used, it should be noted that slight differences in cytokine profiles or activation response may be observed if using primary microglia or other immortalized cell lines. Therefore, the end user can adapt this model to fully characterize the cytokine profile released by NP-activated microglia using either ELISAs or arrays. Future applications of this technique include utilizing primary cell cultures and testing other environmental nanoparticles including those generated from manufacturing, internal combustion engines, aircraft turbine engines, and coal based power generation plants 28-30.

Environmental pollutions, specifically those of the nanoparticle (NP) range (1 - 20 nm diameter), have been linked to obesity and other neurodegenerative diseases due to the ability to cross the blood brain barrier 1-3. Elevated exposure to pollution may induce inflammation in the central nervous system including the hypothalamus 1. One potential mechanism in which this occurs could be through nanoparticle induced activation of microglia (brain immune cells) 4. Prior studies have used in vivo models to study the effects of NPs on brain health which are time-consuming, expensive, and do not directly answer the question of how NPs influence microglia. Microglia play a multifaceted role in the central nervous system, including maintenance of the brain microenvironment and communicating with surrounding neurons via the release of secreted factors and cytokines. Depending on the stimuli, microglia can be activated to an M1 pro-inflammatory or an M2 anti-inflammatory state. For example, M1 activated microglia release pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-&#945;), while M2 activated microglia release anti-inflammatory cytokines including interleukin-4 (IL-4). To validate our surrogate in vitro biosensor for determining neurotoxicity of air pollutants, we measured microglial response to 20 nm silver nanoparticles (AgNPs). The goal of this article is to describe how an in vitro microglial cell line can be used as a surrogate biosensor marker for testing murine microglial response to NPs and how microglial activation affects hypothalamic cells. The long-term intended application of this validated model is to test effects of real-world pollutants on brain health and neurodegenerative disease. We provide a detailed description of an in vitro 96-well format assay for measuring microglial activation and hypothalamic cell survival following the exposure of microglial conditioned media.
Microglial activation was determined following AgNP exposure using a TNF-&#945; enzyme linked immunosorbent assay (ELISA). To determine the effect of activated microglia on hypothalamic cells, the AgNPs were removed from microglial supernatant (conditioned media) using a filtration device. The filtration device retains cytokines while excluding the AgNPs based on size. Briefly, supernatant from microglia treated with or without AgNPs was collected, added to the filters, and centrifuged at 14,000 x g for 15 min. We were then able to determine the influence of microglial secreted cytokines on hypothalamic cell viability. Cell toxicity following exposure to conditioned media (containing cytokines) was determined via a resazurin-based assay as previously described 5,6. Metabolically active cells reduce resazurin and produce a fluorescent signal proportional to the number of viable cells 7.
There are several advantages of using this technique over others (such as co-culture, trans-well setups, or in vivo experiments). Our model provides the ability to directly activate microglia and determine if secreted factors are toxic to neurons 8. The current protocol uses immortalized BV2 microglia stimulated with 20 nm diameter nanoparticles, and immortalized murine hypothalamic cells (designated mHypo-A1/2) 9 for determination of subsequent response. While this protocol has been optimized for these specific conditions, the methods can be altered to be used in other models of microglial-induced cell death, or with other cell types including primary microglia and neurons.

<table><tbody><tr><td>&lt;strong&gt;Cells/Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse microglial cell line (BV2)</td><td>Interlab Cell Line Collection (Genoa, Italy)</td><td>ATL03001</td><td></td></tr><tr><td>Adult Mouse Hypothalamus Cell Line mHypoA-1/2&amp;nbsp;</td><td>Cellutions Biosystems Inc.</td><td>CLU172</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium</td><td>Invitrogen</td><td>10313-039</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAA Labs</td><td>A15-751</td><td></td></tr><tr><td>Penicillin/Streptomycin/Neomycin</td><td>Thermo Fisher Scientific</td><td>15640-055</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Silver nanoparticles (20 nm)</td><td>Sigma-Aldrich</td><td>730793</td><td></td></tr><tr><td>&lt;br/&gt; PrestoBlue Cell Viability Reagent</td><td>Invitrogen</td><td>A13262</td><td></td></tr><tr><td>Mouse TNF-&amp;alpha; ELISA Max Delux</td><td>Biolegend</td><td>430904</td><td></td></tr><tr><td>Lipopolysaccharide</td><td>Sigma-Aldrich</td><td>L4391</td><td></td></tr><tr><td>Sodium Citrate</td><td>Sigma-Aldrich</td><td>S4641</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96 W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td><td>Thermo Fisher Scientific</td><td>165305</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane</td><td>EMD Millipore</td><td>UFC501008</td><td></td></tr><tr><td>SpectraMax M5 Multi-Mode Microplate</td><td>Molecular Devices</td><td>M5</td><td></td></tr><tr><td>Falcon 50 ml Conical Centrifuge Tubes</td><td>Corning, Inc</td><td>&lt;br/&gt; 14-432-22</td><td></td></tr><tr><td>Falcon Cell Strainers 70 &amp;mu;m</td><td>Corning, Inc</td><td>08-771-2</td><td></td></tr><tr><td>Tabletop centrifuge 5430</td><td>Eppendorf</td><td>22620560</td><td></td></tr></tbody></table>

microglia as a surrogate biosensor to determine nanoparticle neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

We show that microglia function as a surrogate biosensor for brain response to nanoparticles using the protocol above. Our results include measurement of the toxic effects of microglial activation on downstream neuronal cell death. Figure 1 demonstrates a workflow of the protocol to activate microglia and determine if secreted cytokines reduce viability of hypothalamic neurons. TNF-&#945; secretion was significantly increased following AgNP exposure (Figure 2). When hypothalamic neurons are exposed to conditioned media from AgNP-activated microglia, cell viability is significantly reduced (Figure 3). Figure 4 outlines the potential mechanism in which AgNPs activate microglia and contribute to hypothalamic cell death.
<img alt="Figure 1" src="/files/ftp_upload/54662/54662fig1.jpg" /> 
Figure 1. Workflow for AgNP Filtration Method. Supernatant from activated microglia is added to filtration devices and centrifuged to remove AgNPs from media. <a href="https://www.jove.com/files/ftp_upload/54662/54662fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54662/54662fig2.jpg" /> 
Figure 2. TNF-&#945; Secretion Is Increased Following AgNP Exposure. Microglial BV2 cells were treated with vehicle control or AgNPs for 2 hr. TNF-&#945; secretion was significantly increased following exposure to AgNPs. Data are presented as mean &#177; SEM. **p &#60; 0.001 vs. control as assessed by Student&#39;s unpaired t-test. <a href="https://www.jove.com/files/ftp_upload/54662/54662fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54662/54662fig3.jpg" /> 
Figure 3. Hypothalamic Cell Viability Is Reduced Following Microglial Conditioned Media Exposure. Hypothalamic cells show increased cell death following exposure to conditioned media from microglia stimulated with AgNP. Data are presented as mean &#177; SEM.*p &#60; 0.05 vs. control as assessed by Student&#39;s unpaired t-test. <a href="https://www.jove.com/files/ftp_upload/54662/54662fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54662/54662fig4.jpg" /> 
Figure 4. Potential Pathway of AgNP Induced Microglial Activation and Influence on Hypothalamic Neurotoxicity. Following exposure to silver nanoparticles, microglia are activated to an M1 pro-inflammatory state, characterized by increased secretion and expression of pro-inflammatory cytokines and makers. The release of the pro-inflammatory cytokines and makers contributes to surrounding neuronal cell death. <a href="https://www.jove.com/files/ftp_upload/54662/54662fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity at JoVE.com

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

microglia-as-surrogate-biosensor-to-determine-nanoparticle

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.9K Views.  Minneapolis Veterans Affairs Health Care System. Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Video: Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Medicine

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

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

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

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

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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

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

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

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Summary

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Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

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

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry