A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High content screening systems allow for a variety of morphological and cytological analysis to be performed simultaneously on many samples. In parallel, in this video, neuron astrocyte co cultures are prepared and seated onto 96 well plates where they are treated with a neurotoxic agent and subjected to various neurotoxicity stains. Plates are loaded into the GE incel analyzer and the investigator software is used for analysis from these plates, readouts for neuro and glial toxicity, such as ne right length neuron count, GFAP expression, an astrocyte area can be obtained.

Hi, I'm Andrew Ball, senior scientist at Millipore Corporation in Temecula, California. Today, Janet Anur, research scientist for high content analysis will demonstrate methods for high content screening of neurotoxicity using neuronal and astrocyte co cultures. This technique employs reagents and protocols from the Neurotox three assay developed here at Millipore to screen for neuro outgrowth, neuronal and astrocyte development and neuronal and astrocyte toxicity.

I hope you enjoy the following sequences. High content screening for neurotoxicity begins by seeding neuron astrocyte co cultures. In 96 well plates, once co cultures have grown to approximately 70 to 80%co fluency detach them.

Using trippin cells should be counted and adjusted to the appropriate density for the assay before they are seeding in the plates. In 90 microliters of growth, medium cells should be allowed to sit on a level surface for at least 15 to 30 minutes to enable even cell distribution, which is critical for this assay. Following this brief incubation growth medium is replaced with 90 microliters, a low serum NGF differentiation.Medium.

Continue to culture these cells until the desired degree of co fluency or differentiation has been obtained and then add 10 microliters of your neurotoxic compound of interest to the 90 microliters of differentiation medium present. The neurotoxic three kit contains the control neurotoxins acrylamide K 2 52 or hydrogen peroxide one. Cells have been exposed to the toxin for the appropriate amount of time.

They can be stained and prepared for imaging, which will show in the next step I.Each reagent kit contains the primary and secondary antibodies and eyes required for the assay, as well as a selection of control compounds. All required buffers are also included in the kit. In this case, we'll be utilizing reagents for the investigation of neurons and astrocytes in a co-culture system at the end of the culture period prior to fixation prewarm the HCS fixation solution at room temperature or 37 degrees Celsius and a chemical fume hood.

Add 100 microliters of fixation solution to each well and allow the cells to be fixed at room temperature for 30 minutes. Remove the fixative solution and dispose of it in accordance with your institution's requirements for disposal of hazardous waste. If cells are to be stained at a later time, rinse them twice with 200 microliters of wash buffer.

Leave the second rinse in the wells tightly sealed and store the plate at four degrees Celsius. If the cells are to be immediately stained, rinse two times with 200 microliters of HCS am immunofluorescence buffer. It's extremely important that cells are not allowed to dry out during staining, so allow the final wash in any series of washes to remain in the wells.

After preparing a working solution of rabbit anti beta three tubulin mouse anti G-F-A-P-H-C-S primary antibodies, remove the immunofluorescence buffer and add 50 microliters of primary incubate at room temperature for one hour. Toward the end of this incubation, a working solution of secondary antibody and hooks to nuclear staining solution can be prepared. This solution should be mixed well and protected from light and stored on ice until use.

Following the one hour incubation, remove the buffer and rinse three times with HCS buffer. Then add 50 microliters of the HCS secondary antibody hooked stain and store room temperature, ensuring that the fixed cells are protected from light. After this one hour incubation, perform two 200 microliter rinses with the HCS immunofluorescence buffer and two 200 microliter rinses with wash buffer.

Seal the plate and image immediately or store the plate at four degrees Celsius until ready to image ensure the plate is protected from light while being stored. For HCS screening, we use the GE Healthcare in cell analyzer 1000. This system must be equipped with beam splitters that can detect fluorescent emission spectra in the blue, green, and red ranges and data acquisition and analysis is performed using investigator software high content screening and analysis.

Using our detection reagents in this instrumentation software package will allow us to generate a series of measurements from each of our stained wells for distinct indices of neurotoxicity. Specifically the indices we will use to quantify the degree of neurotoxicity for each compound. R average neuro length to neuron neuron count, GFAP, staining intensity, astrocyte area, and astrocyte count.

Again, this data is acquired from the fluorescence, images generated from each well and are averaged across all multiple wells for extremely high throughput analysis. Image acquisition setup occurs in the acquisition protocol manager where one can create a sample specific imaging protocol appropriate for the desired magnification, fluorescence, wavelengths, and number of fields to be imaged per well. In this example, we'll be imaging three fluorescent wavelengths, one for each of our markers, the hooks to nuclear counter stain, the neuronal beta three tubulin and the astrocytic glio fibrillary acidic protein or GFAP.

We'll then select our magnification 20 times in this case and our plate brand, each model of which is associated with specifications necessary for proper imaging alignment. Next, we select which wells we want to image. This can either be a subset or the entire plate.

At this point, we can also adjust camera settings. Next, we select the excitation and emission filters appropriate for our fluorescent markers. In this case hst, FITC, and SI three.

Here we can also optimize our exposure times and focus for each fluorescence channel. We can adjust settings to diminish any blurring across the field of view to obtain the sharpest images possible. In the acquisition options window, we can select how many fields we want to image per well and where we'd like those images to be located.

This can range from just a few images per well to many, and they can be located centrally and specific positions randomly or even set to exclude well areas where cells might have been disrupted due to pipetting during staining. At this point, once you're satisfied with all your settings, you're ready to acquire an image set for your plate during acquisition. You can monitor which well is being imaged, which field of view and which wavelength the instrument images each wavelength sequentially, and you can watch each image appear in real time.

In this layout, we have our hooks to nuclear stain appearing in the top panel, the neuronal beta three tubulin in the middle panel and the astrocytic GFAP at the bottom. Neurons tend to have small cell bodies and numerous long thin neurite extensions. Astrocytes tend to have larger cell bodies and may either be spread out to cover a large area or may display projections to give them a more starlike appearance.

These are some example overlaid or merged images of the wells just acquired hooks. Stain cell nuclei are colored blue neuronal cell bodies and neurites expressing beta three tubulin are colored green and astrocytes expressing GFAP are colored. Red image analysis is performed using incel analyzer workstation software.

The software comes with several pre-programmed analysis algorithms. One example is a neite outgrowth detection parameters are rendered to help the software know typical sample dimensions such as nuclear area, cell, body size, and neite length. These values will enable image segmentation.

You can also choose measurements of interest for your assay, including total near right length, near right count or number of nuclei. Data can be exported in multiple formats, including as Excel spreadsheets. Once a protocol is entered, image analysis is performed automatically.

Just observing the beta three tubulin fluorescence channel, you can see round nuclei being outlined in green neuronal cell bodies being outlined in dark blue and long thin lytes also being outlined in green measurements are also displayed as they are generated. Another powerful workstation algorithm is the multi targett analysis protocol. There are a variety of nuclear and cytoplasmic measures that may be obtained.

This analysis can be useful for segmentation of astrocytes and is even capable of distinguishing between cells and co-culture that are positive for GFAP expression outlined in green or negative outlined in red. Here you can see representative imaging samples of a primary rat, hippocampal, astrocyte, and neuronal cultures generated by sequential seeding, seeding astrocytes first, then neurons on top of the glial layer. This is a fused image showing a hooked HCS nuclear stain, the beta three tubulin and GFAP staining.

Separate analysis of the beta three tubulin fluorescence channel allows for neurite outgrowth segmentation where cell bodies are outlined in blue and near rights are outlined in green. Here is a dose response curve illustrating the relationship between neurite length and acrylamide concentration and a primary rat hippocampal monoculture. One can see that as the acrylamide concentration increases above one millimolar, the average near right outgrowth abruptly decreases.

Interestingly, astrocyte and neuronal coal cultures are more resistant to the acrylamide treatment than monocultures. Here is a look at the effects of hydrogen peroxide treatment on neuron count. In our coal cultures taken using 10 fields per well and averaged across all wells, neuron count drastically drops 50%upon increasing the hydrogen peroxide concentration.

In this graph, GFAP intensity is plotted against the acrylamide concentration. GFAP expression is a known indicator of neurotoxicity, and in this graph increases as the concentration of acrylamide is elevated above one milli molar. Interestingly, this is the same concentration in which neuron count drastically drops.

The effects of acrylamide exposure on astrocyte area is demonstrated by this plot, which compares the average astrocyte area in square microns on the Y axis with the concentration of acrylamide on the x axis. Astrocyte count can also be determined using this method. All of this data can be extracted from single stain specimens across multiple wells on the plate.

We prepared earlier. We've just shown you how high content analysis can be used to perform reliable and sensitive assessment of toxicity in neuronal and astrocyte co cultures. We've reviewed the cell handling and immunostaining techniques.

We've also looked at some of the representative data showing the kinds of analysis used to assay for neurotoxicity. When performing this procedure, it's important to ensure that fixed cells are not allowed to dry out during staining and also to ensure that secondary antibody stain cells are protected from light. So that's it.

Thanks for watching and good luck with high content screening.