Een hoog gehalte Imaging Assay voor Identificatie van botulineneurotoxine Remmers

The overall goal of the following experiment is to develop a phenotypic screening assay that IES botulinum mediated cleavage of its substrate SNAP 25 with a view towards identifying small molecules that inhibit toxin function during normal nerve signaling. The action potential causes SNAP 25 to bind the synaptic vesicle to the plasma membrane of the motor neuron terminal resulting in the release of neurotransmitter. However, when botulinum neurotoxin is present, it is taken up by endocytosis and released in the synaptic butone where it cleaves SNAP 25 and so interferes with the release of neurotransmitter causing paralysis.

This assay identifies molecules that can inhibit botulinum toxin activity and thus prevent SNAP 25 cleavage in the presence of botulinum toxin and so restore neurotransmitter release in the neuromuscular junction. A high content imaging assay performed with mouse motor neurons is used to demonstrate the preservation of SNAP 25 resulting in the identification of small molecules that can inhibit the metallo protease activity of botulinum neurotoxin. A.The main advantage of this technology or existing methods like cell-free biochemical assay, is that multiple phenotypic endpoints can be captured simultaneously from a single well containing motor neurons treated with screening compounds and toxin.

This method can help answer key questions encounter during library screening, such as quantification of cell-based activity and cytotoxicity. The implications of this technique extend towards therapy for botulinum intoxication because it can identify inhibitors that act either directly by blocking its metal protease activity or indirectly through prevention of toxin uptake processing, or possibly other mechanisms. Generally, individuals new to this method will struggle because the optimization of immunostaining conditions necessary for SNAP 25 detection can be challenging.

Visual demonstration of this method is critical as the immunostaining step is difficult to perfect as a motor around themselves are rather delicate. The standing and washing steps are the key to developing high quality images required for downstream analysis. All studies that utilize live cells and active toxin are performed under biosafety level two conditions, decontaminate the working areas both before and after these experiments with a 5%detergent disinfectant cleaner, such as micro chem solution.

After differentiating mouse embryonic stem cells into motor neurons as described in the text protocol, use a 12 channel manual pipette to replace the old media. In the 96 well plates with 80 microliters of fresh terminal differentiation media carry out the aspiration and dispensing steps row by row to minimize the exposure of the cells to the ambient air. Next, using a 12 channel pipette, add 10 microliters of 10 x of the test compounds from the source plate, then aspirate twice to mix x.

Treat two columns of six wells with 10 X-D-M-S-O diluted in the media to serve as high signal and low signal controls. Incubate the cells with the compounds for 30 minutes at the seven degrees Celsius and 6%carbon dioxide. Next, dilute the botulinum neurotoxin A in PBS with terminal differentiation media to yield a final 10 X working stock using a multi-channel pipette, add 10 microliters of 10 x botulinum neurotoxin a stock to the wells designated for treatment and also to the low signal control wells.

Treat the wells designated as high control with media alone, decontaminate all plastic wear and other disposables that come in contact with botulinum neurotoxin. A by immersion in 0.825%hypochlorite solution in water for at least 20 minutes. Label the plates clearly to denote toxin use and incubate the plates for four hours at 37 degrees Celsius and 6%carbon dioxide after incubation.

Use a multi-channel pipette to remove the media containing the toxin and compounds from each well and discard it in a 10%bleach solution. Then add 100 microliters of ice cold methanol directly to each well to stop the botulinum neurotoxin. A proteolytic cleavage.

Next, incubate the plates for 15 minutes at room temperature to allow fixation to occur. After fixation, handle the discarded neutralized reagents with biosafety level one protocols and discard accordingly. After discarding the methanol, wash the cells three times with 100 microliters of PBS to rehydrate them prior to immuno staining.

The immuno staining done in a class two biosafety cabinet is performed using an automated workstation arranged to enable reagent access as needed and without human intervention. Load the plates containing the fixed cells into the PLATE magazine and transfer it to the deck as needed for liquid handling operations. Using the 96 pipette head fitted with 250 microliter tips, the workstation aspirates PBS from the wells down to 10 microliters.

The workstation dispenses 100 microliters of blocking buffer containing 1%horse serum and 0.1%tween 20 in PBS into all micro plates. Then return the plates to the plate magazine for a one hour incubation at room temperature. After incubation, remove the blocking buffer and dispense 50 microliters of four micrograms per milliliter of BS antibody and 0.5 micrograms per milliliter of beta three tubulin in blocking buffer into all plates.

Incubate the plates for one hour at room temperature and then remove the primary antibodies and wash three times with 100 microliters of PBST. Prepare two micrograms per milliliter, dilutions of secondary antibodies labeled with different fluoro fours as described in the text protocol. Dispense 50 microliters of the mixture into each well and incubate for one hour at room temperature After incubation, aspirate the secondary antibodies and wash three times with 100 microliters of PBST.

Next at 100 microliters of PBS containing 32 micromolar hooks dye to stain the DNA for nuclei detection. Finally seal the plates with a light impermeable film to protect the fluoro fours from photobleaching. The plates can be stored at four degrees Celsius for weeks without significant loss of signal.

In the image acquisition system, specify the plate type as grinder u. Clear the layout as 96. Well format the number of fields as 16 and the objective as 20 x water.

Then select the channels for nucleus excitation of 405 nanometers. Select channel four for cytoplasm excitation of 640 nanometers. Select channel two for antigen specific antibody channel excitation of 561 nanometers select channel three.

Next, set up the excitation power of each laser. The experiment will have two exposures set exposure, one to one excitation at 640 nanometers and set exposure two to two excitations at 405 nanometers and 561 nanometers. Select Benning as bin two.

Use the high control wells for exposure optimization with maximum intensity as SNAP 25 channel and use the low control wells for minimal intensity. Adjust the exposure so that the intensity of each channel is about half of the saturation level or about 2000 to 2, 500 relative units. Next, identify the optimal Z plane on the cell mask channel using the delta correction script.

In the image analysis software, select the neurite algorithm to segment the neurites. Identify neurite regions based on the secondary objects, the neurites, using a minus three pixel expansion of the beta three tubulin channel as mask. Calculate the intensity of the signal channels for the neurite region and beta three tubulin channel.

Select the desired parameters for export such as neurite length, mean intensities of SNAP 25 beta three, tubulin nuclei number and neurite segment number. With the setup complete. Transfer the plates from the plate stackers using the robot, load them into the high content imager and begin image acquisition.

Shown here is box plot analysis of the percent cleavage calculated from control wells. The pink represents the high signal control that is the wells with just media and no toxin. Very little cleavage is observed in these wells.

The blue represents the low signal control that is the wells with just toxin and no sample compound. These wells registered significant cleavage. These histogram distributions of the control values demonstrate the distance between the two control populations.

The goal of the screening process is to find compounds within the sample population with values closer to the positive control population. The distribution of 192 compound treated samples, which also contained botulinum neurotoxin A is shown here in green. Most of these wells showed significant cleavage.

However, there were a few statistically significant outliers that showed no significant cleavage when compared to controls. The control values are again shown by the pink and blue bars in this histogram distribution. The green bars showed the overlap of the sample values with the control values.

Sample values deemed as hits were selected from the outliers that have percent cleavage values lower than three standard deviations from the median of the sample population. These four hits have similar values to the high signal control and represent the same compound, which is a known botulinum neurotoxin, A inhibitor to synanon that blocks SNAP 25 cleavage. This inhibitor demonstrated robust SNAP 25 protection against botulinum neurotoxin A as wells with both the toxin and this inhibitor behaved similarly to wells with no toxin and dissimilarly to wells with toxin, but without the inhibitor Once mastered.

This technique can be done in four to six hours if it is performed properly. While attempting this procedure, it's important to remember to include multiple controls in order to calculate the statistical performance of the essay and enable a pass fail decision for each individual screening plate Following this procedure. Other methods such as biochemical proteolytic assays can be performed to answer additional questions such as an assessment of screening hits as either direct or indirect inhibitors of Bot.

After watching this video, you should have a good idea of how to automate a phenotypic screening assay that uses quantification of SNAP 25 via immuno staining as its primary endpoint. Don't forget that working with botulinum neurotoxins can be extremely hazardous and precautions such as using personal protection equipment and implementing the appropriate decontamination and disposal procedures for botin. Neurotoxin should always be taken while performing this procedure.