A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Plate 20,000 differentiated mouse ES cells (MES) / well in a 96 well Poly-D lysine coated plates and maintain in motor neuron terminal differentiation media for 5-7 days.

1. Compound Administration and Intoxication with BoNT/A

Perform all of the following work in a BSL2 enclosure to maintain compliance with CDC/NIH guidelines.

1. Prepare 10 mM stock solutions of each library compound in 100% DMSO in polypropylene 96 well plates. Use the 10 mM stock to prepare an intermediate dilution plate that is 10-fold greater than the desired screening concentration. Prepare 100 uM intermediate plate by dispensing 10 mM stock into 96-well plate and dilute it to 100 uM using culture media. Dispense 10 µl of the compounds on to 90 µl of media on the cells¹¹. Test the compounds at 10 uM final concentration and ensure the final percentage of DMSO does not exceed 0.5%.
2. Perform all studies that utilize live cells and active toxin under biosafety level 2 conditions. Replace the old media in the 96-well plates with 80 µl of fresh terminal differentiation media using a 12 channel manual pipette. Carry out aspiration and dispense steps by rows to avoid exposure of the cells without media to the ambient air.
3. Pretreat cells with compounds prior to application of toxin by addition of 10 µl of 10x compounds from the source plate using a 12 channel pipette, followed by mixing by aspiration (twice). For each 96-well plate, treat two columns of 6 wells with 10x DMSO diluted in the media to serve as high signal and low signal controls. The column without BoNT treatment exhibits the highest level of the full length SNAP-25 (high signal control). Treat the other column with BoNT alone (without any compounds) as the low signal control. Use low control to observe the efficiency of BoNT cleavage of SNAP-25 and its detection with the BACS antibody (Figure 2).
4. Incubate cells with compounds for 30 min at 37 °C and 6% CO₂ in the cell culture incubator.
5. Prepare Botulinum neurotoxin A from a 1 mg/ml commercial source in Phosphate buffered saline (PBS) to a final 10x working stock by diluting with terminal differentiation media.
6. Initiate intoxication by adding 10 µl of 10x BoNT/A stock into wells designated for treatment and low signal controls using a multichannel pipette (Figure 2). Treat the wells designated as high control with media alone.
7. Decontaminate all plastic ware and other disposables that comes in contact with BoNT/A during the study by immersion in 0.825% hypochlorite solution in water for at least 20 min. Perform all procedures in biosafety cabinets and decontaminate working areas both before and after experiments with 5% detergent disinfectant cleaner such as MicroChem solution.
8. Label the plates clearly to denote toxin use and incubate plates treated with 1 nM/L BoNT/A for 4 hr at 37 °C and 6% CO₂ in a cell culture incubator. Display appropriate signage to inform colleagues that toxin is in use in the laboratory.
9. Stop BONT/A proteolytic cleavage by methanol fixation. Remove media containing toxin and compounds from each well with a multichannel pipette and discard in 10% bleach solution. Add ice cold methanol (100 µl) directly to each well.
10. Incubate plates for 15 min at room temperature (RT) to allow fixation to occur.
11. After fixation, safely handle the discarded reagents (considered neutralized) with biosafety level 1 protocols and discard accordingly.
12. Discard methanol and use 100 µl of PBS to wash the cells and rehydrate them prior to immunostaining. Perform two additional PBS washes.
13. After the final wash, leave PBS in the wells, seal plates with microplate adhesive film and store at 4 °C until analysis. Store plates at 4 °C for several days.

2. Immunostaining

The immunostaining procedure is a labor-intensive, multistep operation that includes repetitive reagent dispense/aspirate cycles and extensive plate washing which can lead to the potential introduction of significant intraplate and interplate variability. A semi-automated approach is applied to save the time of laboratory personnel, increase assay throughput, and minimize immunostaining variability.

1. Use an automated workstation equipped with a 96-well head capable of transferring volumes up to 250 µl and integrated with a robotic gripper arm to move labware into different locations on the modular deck (see Material and Equipment) for this protocol.
   1. The modular deck is designed to host different types of labware and can support up to 11 items at any one time. The automated microplate handler is equipped with a dual magazine microplate stacker in order to hold and manipulate up to 50 plates per cycle.
   2. The automated workstation is located inside of a Class II biosafety cabinet designed for laboratory automation equipment to provide sterility and safety. For automated immunostaining, the deck is arranged as shown in Figure 3 to enable reagent access as needed and without human intervention.
2. Pre-load plates containing fixed cells into the plate magazine and transfer to the deck as needed for liquid handling operations. Use the 96 pipette head fitted with 250 µl tips to aspirate PBS from the wells down to 10 µl. Permeabilize cellular membranes to facilitate antibody binding to intracellular targets with permeabilization buffer (0.1% Triton-X 100). Dispense permeabilization buffer (100 µl) into each well before returning the plates to the second storage magazine of the microplate stacker for a 15 min incubation at room temperature (RT).
3. Remove permeabilization buffer and perform plate washing with PBS (twice) as previously described in step 1.13.
4. After the last wash step, remove the PBS buffer and dispense 100 µl of blocking buffer containing 1% horse serum and 0.1% Tween 20 in PBS into all microplates before returning them to the plate magazine for 1hr incubation at RT.
5. Use two primary antibodies for immunostaining: a rabbit polyclonal anti-mouse BACS antibody, for detection of full length SNAP-25 and a mouse monoclonal anti-III tubulin antibody for detection of neurons (see Material and Equipment). After 60 min of incubation, remove blocking buffer and dispense 50 µl of 4ug/ml of BACS antibody and 0.5 µg/ml of III-tubulin in blocking buffer into all plates.
6. Incubate for 1hr at RT remove the primary antibodies and wash 3 times with 100 µl of PBS containing 0.05% Tween (PBST).
7. Use an anti-mouse IgG labeled with Alexa Fluor 647 to visualize the -III tubulin and anti-rabbit IgG tagged with Alexa Fluor 568 to visualize the BACS antibody. Prepare both antibodies as a 2 µg/ml dilution in blocking buffer and dispense 50 µl of the mixture into each well.
   NOTE: The secondary antibodies selected for immunostaining are labeled with different fluorophores to allow detection of their emitted fluorescent light at distinct wavelengths using different channels within the detector of the High Content Imager.
8. Incubate for 1 hr at RT. Aspirate the secondary antibodies and wash 3 times with 100 µl of PBST.
9. After the final wash, add 100 µl of PBS containing Hoechst dye (32 µM) to stain the DNA for nuclei detection.
10. Seal the plates with a light impermeable film to protect the fluorophores from photobleaching. Plates can be stored at 4 °C for weeks without significant loss of signal.

3. Imaging

NOTE: Perform image acquisition using High Content Imaging System (See Materials and Equipment).

1. Specification of High Content Imager Definitions:
   1. Select Plate type, layout, number of fields, objective: Greiner u clear, 96 well format, 16, 20X water respectively.
   2. Select channels: nucleus excitation EX: 405 nm as channel 4; cytoplasm EX: 644 nm as channel 2; antigen specific antibody channel EX: 561 nm as channel 3 and Set up excitation power of each laser, Setup experiment as 2 exposures, Exposure 1 with one excitation at 644 nm, Exposure 2 with two excitations at 405 nm and 561 nm. Select binning as Bin2.
   3. Use high control wells for exposure optimization with maximum intensity as SNAP-25 channel and low control wells for minimal intensity.
   4. Adjust the exposure, so that the intensity of each channel is about half of the saturation level (2,000-2,500 relative units).
   5. Identify the optimal Z plane on cell mask channel using delta correction script. See Figure 5 for results after immunostaining and imaging. Export data to the server where the image analysis software is residing.

4. Image Analysis (Figure 6)

NOTE: The following steps describe application of the Columbus software algorithms.

1. Select an appropriate algorithm to segment the primary objects (nuclei). If required, adjust background threshold and contrast parameters. The Columbus software provides 4 nuclei detection methods. Select the method that segment nuclei accurately by visually inspecting segmented nuclei (in Figure 6a method M is selected).
2. Select the neurite algorithm (CSIRO Neurite Analysis 2) to segment the neurites If required, adjust background threshold and contrast parameters to remove background. See Figure 6b for parameters used to detect neurites.
3. Identify the neurite regions (neurite segments) based on secondary objects (neurites) using a -3 pixel expansion of the β-III tubulin channel (Alexa Flour 640) as mask (Figure 6c).
4. Calculate intensity of the signal channels (SNAP-25, (Alexa Flour 568) for the neurites region and β-III tubulin channel (Figure 6d & 6e).
5. Select desired parameters for export, such as neurite length, mean intensities of SNAP-25, β-III tubulin, nuclei number, neurite segment number, etc. (Figure 6f).
6. Transfer plates from plate stackers using the robot and load into the High Content Imager for image acquisition.

5. Data Analysis:

To assess the robustness of the designed assay, calculate the following parameters from the plate-based experiment.

1. Signal (S) to background (B):S/B = mean intensity of high signal control) / mean intensity of low signal control
2. Coefficient of Variation (CV) of signal and background (to measure the uniformity of the specific signal): CV = (Standard Deviation (SD) / mean of sample)*100(%), to determine the variability in liquid handling, reagents, etc.
3. Signal to Noise: S = (mean intensity of signal – mean intensity of low signal control) / SD of low control
   1. Calculate the Z’ factor with intoxication of one half of a 96-well plate and a mock intoxication of the other half. Z’ = 1 – 3 * (SD of high signal control + SD of low signal control) / (mean of high signal control – mean of low signal control). For the further analysis of the screening data, normalize some of the primary raw parameters on a plate basis to allow comparison between plates and between different days of experiments.
4. Percent of cleavage, reflecting the reduction of the signal associated with full length SNAP-25 detected by specific antibody (BACS) : % Cleavage = 100% * (mean intensity of high signal control – intensity of sample) / mean intensity of high signal control.
5. Percent of Inhibition of cleavage: % Inhibition = 100% * (intensity of sample – mean intensity of low signal control) / (mean intensity of high signal control – mean intensity of low signal control).