The overall goal of this protocol is to describe an in vitro surrogate biosensor for testing microglial response to nanoparticles. We will then determine neurotoxicity following exposure to conditioned media from microglia stimulated with nanoparticles. This method can help answer key questions in the neuroscience field such as how actively screened nanoparticles for neurotoxicity.
The main advantage of this technique is that our model provides the ability to directly activate microglia and determine if secretive factors are neurotoxins without the use of expensive long term rodent studies. Though this method can provide insight into microglial induced cell death following microparticle exposure. It can also be applied to other models of neurodegenerative diseases within the context of testing potential neurotoxic environmental pollutants.
Generally individuals new to this method will struggle with the condition media filtration step. Because the filtration efficiency may differ depending on the size, shape, and the concentration of the nanoparticles. In preparation warm the DMEM supplemented with 10%FBS and 1%PSN to 37 degrees Celsius.
Then take frozen BV2 microglial cells obtained from passages 18 25, and rapidly thaw them at 37 degrees Celsius. Next, gently transfer the thawed cells to a 75 square-centimeter vented flask containing 10 milliliters of medium and incubate the flask. After 24 hours replace the medium and continue growing the cells until they reach a confluency of 70-80%At this point aspirate the medium and add 500 microliters of warm 1 ex trypsin EDTA solution.
After allowing the flask to incubate for a few more minutes use a scraper to collect the cells into 5 milliliters of warm DMEM. Then filter the cells from the flask using a 70 micron strainer. Repeat this filtration three times using the same strainer and collect the cells in a 50 milliliter tube.
Once filtered, count the cells and then seed a black-walled clear bottom plate with 8000 cells per well in 200 microliters of supplemented DMEM. After 24 hours of incubation, replace the medium in the wells with serum-free DMEM to starve the microglia. After 24 more hours of incubation, proceed to activate the microglia.
To do so, first dilute silver nanoparticles in serum-free DMEM to the desired working concentration. Then replace 100 microliters of medium with the treatment compound. Be sure to include a negative control of diluted vehicle solution without the nanoparticles.
Now continue the incubation for 24 hours and proceed with the analysis. To begin collect 200 microliters of supernatant medium from each plate well and load it onto a filter that will remove the nanoparticles placed over a micro centrifuge tube. It's important to remember that when filtering the nanoparticles their size, shape, and the concentration are a factor for fusion efficiency.
So it is advisable to test the filtration beforehand. Now spin the tubes down at 14000g at room temperature for 15 minutes. Then discard the flow-through which contains the nanoparticles and place the filters upside down in new collection tubes.
Spin the filters upside down at 1000g for two minutes to collect concentrated conditioned medium containing cytokines. Next increase the volume of the collection to 400 microliters with freshly made supplemental DMEM and store the tubes on ice. To ensure residual sulfur nanoparticles are removed from filtered media, prepare unfiltered nanoparticle standards at a range of concentration from a blank to 0.2 micrograms per milliliter in serum-free DMEM.
Then aliquot 50 milliliters of the filtered samples and of each standard into a black-walled clear bottom plate and measure absorbance from 390 to 410 nanometers. To measure the concentration of TNF-alpha in the samples, use half their remaining volume in a commercially available kit. For this assay establish hypothalamic cells from frozen stocks.
Using the same methods described for establishing microglial cultures. Ultimately use them to seed black-walled clear bottom plates with 5000 cells per well in 200 microliters of supplemented DMEM. After 24 hours of culturing, remove 100 microliters of medium and replace it with 100 microliters of filtered and concentrated medium collected from the activated microglial cell cultures then incubate the plate for 24 hours under standard conditions.
The next day add 22 microliters of resazurin reagent to each well and continue the incubation for 20 minutes. Then with a mulit-mode spectrophotometer take a florescence measure to estimate the cells'viability in terms of relative fluorescence units. With the outlined protocol microglia function can be used as a surrogate biosensor for the brain's response to nanoparticles.
TNF-alfa secretion by microglia was significantly increased following sliver nanoparticle exposure. When hypothalamic neurons were exposed to conditioned medium from the silver nanoparticle activated microglia, the viability of the neurons was significantly reduced. In principle exposure to the silver nanoparticles activated the microglia to an M1 pro-inflammatory state.
Then the released pro-inflammatory cytokines contribute to surrounding cell death. After watching this video you should have a good understanding of how to use microglia as a surrogate biosensor for testing response to nanoparticles and if this response is neurotoxic. Once mastered, this technique can be done in three or four days if it's performed properly.
Following this procedure, other methods such as profiling other secreted cytokines following microglial activation can be performed in order to phenotype microglial activation states.
