Automated high-content imaging in iPSC-derived neuronal progenitors

Induced pluripotent stem cells (iPSCs) have great potential as physiological disease models for human disorders where access to primary cells is difficult, such as neurons. In recent years, many protocols have been developed for the generation of iPSCs and the differentiation into specialised cell subtypes of interest. More recently, these models have been modified to allow large-scale phenotyping and high-content screening of small molecule compounds in iPSC-derived neuronal cells. Here, we describe the automated seeding of day 11 ventral midbrain progenitor cells into 96-well plates, administration of compounds, automated staining for immunofluorescence, the acquisition of images on a high-content screening platform and workflows for image analysis.


Introduction
High-Content Screening (HCS) is a powerful technique to investigate gene function in an unbiased manner within the cellular context [1][2][3] .Abilities to a) multiplex readouts for multi-parametric analysis and b) cell painting in combination with enhanced machine learning for image analysis have further pushed HCS to become an unrivalled technology for discoveries of cell-based phenotypes and gene/protein networks [4][5][6][7][8][9] .This is particularly true for discoveries that aim to determine disease mechanisms or the identification of novel drug targets.
Historically, HCS has relied on easy-to-transfect cancer cell lines.More recently, a shift towards the use of primary cells and more physiologically relevant model systems has been made feasible using patient-derived cells in multi-well microplates and a two-or threedimensional format [ 10 , 11 ].For some time, the use of non-dividing neuronal cells has remained a challenge in HCS, due to the difficulties in transfection of reporter genes and the inherent variability in cell behaviour and phenotypes.The advent of induced pluripotent stem cells [12] (iPSCs) and their differentiation into a wide range of cell lineages has allowed the study of human cells from tissues that were otherwise not easily accessible, such as the brain or the heart [13] .Over the years, protocols have emerged for the use of iPSC-derived neurons in high-throughput phenotyping and highcontent imaging [14][15][16] .However, the number of screens performed in such cell types remain low and the setup of standardized workflows for generation and high-content screening are still technically challenging.
Here, we describe a protocol for high-content screening of small molecule libraries in patient-derived neural cells and discuss potential pitfalls and challenges.We used iPSCs generated from skin fibroblasts and then applied a differentiation protocol to develop these into midbrain dopaminergic (mDA) neurons [17][18][19] .Day 11 ventral midbrain progenitor cells were seeded onto 96-well microplates and treated with a small molecule compound library.The cells were stained with antibodies against the autophagy marker LC3 and imaged on a PerkinElmer Opera Phenix high-content imaging platform in a semi-automated manner.Finally, an image analysis pipeline was developed to count the number of objects (autophagosomes) in these cells.
For this protocol to be implemented, fully characterized, truly pluripotent patient-derived and age-matched control iPSC lines are required.Phenotypic assay set up must take place on iPSC-derived neurons prior to the drug screen, looking for assays that distinguish patients from controls in a high content immunofluorescence setting; the subsequent screen will aim to identify compound 'hits' that restore or ameliorate this patient-specific phenotype.One main limiting factor in iPSC technology is line variability but, especially in monogenic disorders, the use of multiple patient lines and appropriate age-matched controls (including isogenic controls generated via CRISPR genome editing) allow for robust phenotypic assay set-up and results in downstream experiments [13] .In the protocol described, an autophagy (LC3)based assay was used for the iPSC-derived neuronal screening; however, the workflow can be adapted to analysis of other proteins of interests.For the drug screening, access to an automated liquid handling system, an acoustic dispenser and a high-content imaging plat-form is required.Finally, for image analysis, bioinformatics support is recommended.

iPSCs
Characterize all iPSC lines to ensure true pluripotency prior to neuronal differentiation and screening experiments.Pluripotency characterization includes: a) expression of pluripotency markers via immunofluorescence and RT-PCR, b) Epi-Pluri-Score [20] (examining DNA-methylation levels at specific CpG sites), c) spontaneous in vitro differentiation into all three germ layers, d) maintenance of genomic integrity after reprogramming (e.g.SNP array analysis to ensure no large pathogenic deletions/ duplications have been acquired, and genomic DNA sequencing of targeted areas to ensure disease-causing mutations are maintained) and e) non-integrating vector (e.g.Sendai virus) clearance, if relevant reprogramming methods have been used [ 17 , 18 ] (Notes 2-5).

Reagents, media, plasticware
Example manufacturers and catalogue numbers are provided below (Note 1).As an alternative to the Opera Phenix microscope, other high content imaging platforms can be used, e.g. the Thermo Scientific CellInsight CX7 Pro High-Content Screening (HCS) Platform.As an alternative to the Echo520 or Echo550 accoustic dispensers, standard liquid handling robots could be used.This may require additional dilution steps to prepare the right stock concentrations.Similarly, other high-content imaging platforms can be used and may require adjustments in the protocol setup (Section 2.3.).As an alternative to IMAGEJ, other image analysis software can be used such as CellProfiler [21] , Icy or commercial software that is often integrated into the hardware of the high-content imaging platform (Section 2.5.).

Procedure
Carry out all experiments at room temperature unless otherwise specified.
Resuspend in culture medium supplemented with thiazovivin (stock concentration 10 mM in DMSO, 10 M final concentration) in ratio of one cryovial per one well of a 6 well plate, and plate on Matrigelor Vitronectin XF-coated 6-well plate.After 24 h, change to iPSCs maintenance medium without thiazovivin (Note 9). 4 Maintain iPSCs in mTeSR1 on 6-well, Tissue Culture-treated, Matrigel-coated plates.Alternatively, use TeSR-E8 on Vitronectin XF-coated plates.Change media daily [ 22 , 23 ] (2 ml per 6-well plate well) and regularly passage (when confluent; approximately every 4-7 days depending on each iPSC line and cell seeding ratios) (Note 10). 5 To passage iPSC lines, wash cells in 1x DPBS (2 ml/ well of a 6well plate) and add 1 ml EDTA 0.02%.After approximately 5 min of incubation at 37 °C, aspirate EDTA.iPSCs should be gently lifted in clumps of 5 to 10 cells using sequential washing of separate sections of the well from low to high, with 1 ml culture media each for each wash, and using a P1000 pipette.Avoid contact of cells with media if not ready to be dissociated, as this would re-accumulate Ca 2 + and thus cell-cell adhesion.Then, transfer to a sterile Falcon tube and, finally, to new Matrigel (or Vitronectin XF)-coated plates.
The ratio of passaging is usually 1 well into 2 wells for cells cultured in the mTESR1/Matrigel system.For TeSR-E8/ Vitronectin XF maintenance, consider ratios of 1 well into 3-4 wells, especially if spontaneous differentiation is prominent (Note 11).6 To remove spontaneously differentiating cells from iPSC cultures, choose from the options below (Note 12): 6A.For iPSCs cultures on Vitronectin-coated plates: after EDTA application, do not dissociate iPSCs with a P1000 pipette.Instead, slowly add 1 ml of medium to each well, and gently tap the sides of the plate to dissociate clumps of undifferentiated iPSCs and allow differentiating cells to remain attached.Leave newly dissociated iPSCs to sediment to the bottom of a 15 ml Falcon tube filled with culture medium for 3-4 min; subsequently, aspirate and discard most of the supernatant (containing differentiating single cells) and only plate the bottom 2 ml (containing undifferentiated iPSC clumps), at a final volume of 2 ml/ well.
6B Use ReLeSR (main method for iPSCs cultured on the matrigel/ mTeSR system).Wash iPSCs with 1x DBPS and apply ReLeSR (1 ml per well of a 6-well plate) for 1 min.Then, aspirate ReLeSR and leave wells to dry out for 4 min at room temperature (optimal time will depend on the cell type; cells are ready when clear sign of cell-cell detaching are visible).Subsequently, dissociate non-differentiating iPSC colonies by adding 1 ml/well of culture medium and gently tapping the plate, then plate onto newly coated plates (see manufacturer protocol).
6C. Use the gelatine 0.1% method.Coat 6-well plates with sterile 0.1% gelatine in Milli-Q water and incubate at 37 °C for 1-24 h.Harvest iPSC lines exhibiting obvious spontaneous differentiation using TrypLE, centrifuge at 300 g for 5 min, re-suspend the pellet in mTeSR1 and Thiazovivin (stock concentration 10 mM in DMSO, diluted 1:1000 in culture medium to a final concentration of 10 μM), seed on the gelatine-coated wells and incubate at 37 °C for 30 min (allowing for differentiating cells to adhere to the bottom of the plate).Subsequently, aspirate the medium (containing un-differentiated iPSCs) and plate onto Matrigelcoated plates.Time of seeding onto gelatine-coated dishes might be extended to maximum 40 min.
1 To freeze and store iPSCs, dissociate cells when confluent with Try-pLE, centrifuge at 300 g for 5 min and resuspend contents of one well of a 6-well plate in 1 ml of 90% KOSR complete medium and 10% DMSO per cryovial.Store vials in a freezing container, e.g., Nal-gene® Mr. Frosty (Sigma Aldrich C1562-1EA) or similar, at − 80 °C for 24 h before transferring to liquid nitrogen.Note: frozen iPSCs can be stored at − 80 °C for a maximum of three months; longer time will severely affect cell survival after thawing.

Differentiation into dopaminergic progenitor cells
1 On Day 0, to form Embryoid Bodies (EBs), harvest iPSCs: aspirate medium, wash with 1x DPBS (2 ml/ well), apply TrypLE (1 ml/ well) and incubate at 37 °C for 5 min.Then, aspirate TrypLE, dissociate with mTeSR1/ TeSR-E8 using a P1000 pipette, transfer to a 15 ml Falcon tube, and centrifuge at 300 g for 5 min.Count the number of total cells harvested, resuspend 8 × 10 6 cells in 11 ml of differentiation medium and plate onto one non-adherent 10 cm Petri dish (ratio: 8E6 cells/10 cm petri dish).Note: if using a smaller petri dish, scale down the number of cells to be plated (e.g. for a 6 cm Petri dish, seed 4 × 10 6 cells).2 Change media every 48 h for the duration of the differentiation.
2B To induce neural identities via dual SMAD inhibition, supplement medium with 100 nM LDN193189 (Bone Morphogenic Pathway inhibitor) for 9 days (day 0 to day 8), and with 10 M SB431542 (TGF beta blocker) for 6 days (day 0 to day 5) [26] .For LDN193189, to minimise pipetting errors, pre-dilute stock solution by 1:100 in neurobasal medium, and then further dilute this by 1:1000 into the final differentiation medium.
2C. Use Sonic hedgehog (SHH-C24II) 100 ng/ml for 9 days (Day 0 to day 8) and the SHH pathway activator Purmorphamine 0.5 M for 7 days (Day 2 to Day 8) to guide the differentiating cells towards floorplate identities [27] .For purmorphamine, to minimise pipetting errors, predilute stock solution by 1:100 in neurobasal medium, and then further dilute this by 1:200 into the final differentiation medium.

Cell seeding and compound administration (
1 On Day 11 of differentiation, dry PO/Fn/LN-coated 96-well low skirted destination microplates.2 Administer compounds of the library of choice to the coated assay plates using the Labcyte Echo 550 Liquid Handler: Library compounds with a stock concentration of 10 mM are located in every second well of a 384 LDV Echo plate.To achieve a final concentration of 10 μM in the 96 well assay plates, transfer 100 nl of the compounds from the 384 LDV plates into the coated 96 well plates using the Labcyte Echo Dose-Response software ( Fig. 3 A).Here one can compress the intermitted pattern of the 384 well source plate by defining every second well in columns 3 to 21 of the source plate and every well in columns 2 to 11 in the destination plate as samples.For a one-to-one transfer provide a single source concentration and transfer volume and select one data point per curve.Then, dispense 100 nl of negative and positive controls.For LC3-based high content screening, use DMSO as negative control in Column 1 and Bafilomycin A1 (stock concentration 10 μM, final concentration 10 nM after adding the cell suspension) as positive control in Column 12 ( Fig. 3 B).If stock solutions of both controls are positioned in the library source plate itself, select the respective wells in the source plate and define column 1 and 12 in the destination plate as controls within the Echo Dose-Response protocol.
1 To test each compound in triplicates, dispense the compounds and controls of one 384 well source plate into three 96-well destination plates.Further automation using integrated stackers, a microplate delidder, and a robotic arm controlled by the Tempo software facilitate the drug dispensing process. 2 To dissociate Day 11 cells from 12-well plates, firstly make KOSR complete medium.Medium composition (500 ml): 390 ml Knockout DMEM, 100 ml Knockout-Serum Replacement (20%), 5 ml lglutamine (2 mM), 5 ml Non-Essential Amino Acids 100x (1%), 500 μl 2-Mercaptoethanol (50 mM). 3 Wash Day 11 cells with 1x DPBS and incubate with 500 l of Accumax for 20 min at 37 °C.Add KOSR complete medium to stop the enzymatic reaction, dissociate cells with a P1000 pipette and pellet by transferring in a KOSR-containing 15 ml Falcon tube and centrifuging at 300 g for 5 min.Re -suspended in Day 11 medium at a dilution of 15,000 cells/ml.Seed 100 l per well (15,000 cells/ well) in the 96-well low skirted destination microplates using the Biotek Multi-Flo Multi-Mode Dispenser.4 Culture cells at 37 °C for 24 h, then fix and stain as described below ( Section 3.4 .).

Automated immunostaining
For automated immunostaining, use a washer and dispenser such as the Biotek EL406.This can be integrated into a larger liquid handler set up, with a Cytomat hotel and the Beckman Biomek NXp liquid handler including an Inheco cooling unit.Set up scripts for the individual dispensing and washing steps and run using the SAMI Ex scheduling software.Move plates to the individual stations in the protocol using a Scara robotic arm ( Tables 2 and 3 ).
Set up the following protocol using the liquid handler pipeline listed below the fixation/staining steps ( Tables 2 and 3 ): 1. Fix with 4% PFA, as described in Table 2 .
2. Permeabilize and fix a second time in ice-cold methanol (kept at − 20 °C) for 15 min at + 4 °C, then wash three times with 1 x DPBS.  2 Automated PFA fixation.The stations the assay plates run through during the fixation process are listed in the left column.The individual steps of the fixation protocol at the respective stations are described in the right column.Wash with 1 x DPBS using the washer head, in 3 cycles:Aspirate at travel rate 1Dispense 100 μl of 1x DPBS at flow rate 1 Cytomat Hotel Home position.Plates will be placed here, ready for subsequent steps of the staining protocol or for collection and intermediate storage 3. Incubate in blocking buffer (1 x DBPS, 10% FBS and 0.1% Triton) for 1 h at room temperature.
5. Incubate cells with secondary antibody (1:400 dilution) in blocking buffer (50 l/well) and DAPI (1:1000) for 1 h at room temperature.Wash three times with 1 x DPBS, then store at + 4 °C, in the dark, until image acquisition.

Image acquisition
1.For imaging, use the PerkinElmer Opera Phenix confocal microscope ( Note 7 ).
2. For image acquisition, use the Harmony software provided with the PerkinElmer Opera Phenix.For the set-up, follow steps below: 2A. Choose the appropriate plate type from the drop-down menu.Ideally choose a low skirted 96 well plate for the objective to be able to move into the far corners of the plate.
2B Choose a two peak autofocus.2C.To obtain high resolution images, use a water objective with high numerical aperture in confocal mode and a binning setting of 1.We obtained good results using the 40x water objective (Aperture 1.1).
2D. Select the appropriate channels for your staining.In the Harmony software, the pre-set Hoechst channel and the Alexa 488 channel can be used.
Hoechst 33,342: 375 nm excitation laser, 435-480 nm emission filter Alexa 488: 488 nm excitation laser and 500-550 nm emission filter 2E.Adopt the pre-set exposure time, laser power and height settings to check individual wells for cells using the "snapshot " function.
2F.One can identify the most suitable plane within the neuronal progenitor cultures by setting up a stack in the "Layout Selection " tab: For one-layered cultures on low skirted plates with a bottom thickness of around 0.18 mm, one can start with a bottom plane of − 5 μm and go up in increments of 0.5 μm to reach a top plane of 5 μm.By switching from the "Define Layout " tab to the "Image " tab, one can now browse through the stack and define the section with highly resolved LC3 spots.Manually transfer the height of the preferred section into your channel setting.
2 G.To determine the most appropriate exposure settings, pick a well containing a positive control in the "Define Layout " tab.Increase power and exposure time to ideally reach a pixel intensity of around 9000 for LC3 spots and nuclei.Pixel intensity of individual spots can be checked during the set up process (right click, choose: show intensity, click on the desired spot).We previously acquired images with the following settings: Hoechst 33,342: time = 300 ms, power = 100%, height = − 1.0 Alexa 488: time = 1000 ms, power = 100%, height = − 1.0 2H.To avoid 'bleed-though' between channels, separate both channels with the "Channel Sequence " option.
2I. Select all wells, and 20 fields per well, to be imaged.3.For further automation a rack for plate storage and a robotic arm for plate feeding into the microscope can be used.When using the PerkinElmer Opera Phenix these will be under the control of the plate::works TM software.Within the plate::works TM software, set up your "method " (the path the individual plate takes through the imaging set up).For fixed and stained assay plates of the LC3 screen, this will be: Rack = > Barcode Reader = > Phenix = > Rack.Define the plate count, experiment name and owner as specified earlier in the Harmony software, as well as duration of imaging.Fill the stack with the defined amount of assay plates.Within the "Run Experiment " window in the Harmony software, switch to the "Remote " control and start the imaging process.
4. Upon completion, export captured data to the Columbus Image Data Storage and Analysis System and, subsequently, as .TIF images to institutional secure drives as required.

Image analysis ( Fig. 1 B)
Process images using a high-content analysis computer equipped with at least 32 GB memory, ideally a Supermicro SuperServer 4048B-TR4FT system with 1 TB memory that we used and a recent ImageJ ver-sion (we used version 1.52) (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2022.).Analyse a full plate of 1920 images (96 wells x 20 field of views) (25 GB) as a set on each ImageJ instance using a custom ImageJ macro with a modified workflow from already published work [30] : 1 Read the image set of a plate into the memory and convert it into a hyperstack.Perform iterations of the calculation as the following: 2 Apply a Mean filter with 20 pixels radius parameter on the nuclear channel (Channel 1: 375 nm) for image pre-processing.3 Segment nuclei using a watershed segmentation algorithm with appropriately optimized parameter that quantifies the nuclei number based on the local maximum pixel intensities.4 Apply a watershed segmentation to the LC3 channel (Channel 2: 488 nm) without any pre-processing steps to quantify the number of LC3 puncta.5 Calculate the LC3 puncta / nuclei ratio per well using an R scri pt R [31] ( https://www.r-project.org/, we used R version 3.5.2). 6 Process the resulting data using the Bioconductor R package cell-HTS2 [32] version 2.46.1.Normalize data using the negative control values and transform to Z scores.7 Calculate the mean value of the 3 replicates for the final, single Z score per treatment ( Note 15 ).

Flow diagram
A schematic summarizing the iPSC neuronal differentiation, seeding of ventral midbrain progenitors onto multiwell plates for compound screening, and image analysis is provided in Fig. 1 .

Troubleshooting/ notes
1 Prepare and store all media and reagents at the temperature specified by the manufacturer.Diligently follow waste disposal regulations when disposing materials.4 When setting up phenotypic assays for subsequent screening, use as many patient-derived and age-matched iPSC lines as possible; in cases of monogenic disorders, CRISPR/Cas9-generated isogenic controls (where the disease-causing mutation is substituted with the 'wild-type' sequence via Homology Directed Repair) should also be considered [ 17 , 33 ].This approach helps overcome the inherent issue of line-to-line variability in iPSC models and ensures robust and reproducible results in downstream experiments (Section C1). 5 Maintain iPSC lines in culture for a maximum of 3 months after thawing (10-15 passages) to avoid acquisition of chromosomal/ genetic and epigenetic abnormalities that might influence results of downstream applications (Section C1).6 Maintenance on mTeSR1/ matrigel or TeSR-E8/ Vitronectin largely depends on the conditions used during iPSC reprogramming.It is generally preferable to continue using the system the iPSCs were generated on; changes from one system to the other are also possible (but might exert stress on the cells and/ or result in unwanted spontaneous differentiation).If matrigel is unavailable or difficult to purchase, substitute with GeltreX, (e.g., A1569601 or A1413302 by ThermoFisher).7 Thaw iPSCs as quickly as possible, at 37 °C, as slower thawing can result in reduced cell survival.Do not wait until the cryovial is fully thawed; instead, start resuspending in culture medium when a small frozen clump can still be seen (Section C1.3.).8 Daily media change when maintaining iPSCs is crucial; it reduces cell stress, and the possibility of iPSCs starting to spontaneously differentiate, and promotes healthy and efficient downstream differentiations.Changing media daily prevents the pH (mainly dependent on confluence) from becoming too acidic, which in turn induces cellular stress.Moreover, maintenance media contain FGF2 which is crucial to maintain pluripotency but degrades very quickly at 37 °C.Stress and reduced FGF2 levels can trigger the release of differentiation factors into the medium (Section C1.4.).9 Passage iPSCs when confluent.For cells cultured on Matrigel, there should be no gaps seen in the wells under microscopy.Conversely, in vitronectin-coated plates, iPSCs often grow in distinct, somewhat circular, colonies that do not expand to cover the whole well (some gaps between colonies are often present).For these, passage when the centre of the iPSC colonies becomes very dense (prolonging culture without passaging will stress the cells and lead to spontaneous differentiation) (Section C1.5.). 10 The method described in 6A. is the preferred, and usually sufficient, method for 'cleaning' iPSCs cultured on vitronectin-coated plates; here, undifferentiated cells form often-distinct colonies that can be detached by gentle tapping after incubation with EDTA.A (usually small) number of differentiating single cells can be seen around the iPSC colonies, which either do not detach with tapping or stay in the supernatant and can be removed by aspiration (with the clumps of iPSCs sedimenting at the bottom of the Falcon tube).Methods 6B. and 6C.can be used for iPSCs cultured on both Matrigel and vitronectin, and sometimes a combination of all methods (and repeated cleaning) might be required.In cases of persistent spontaneous differentiation, have low threshold of discarding the cells and thawing a new cryovial (Section C1.6.).11 Number of starting cells for downstream mDA neuronal differentiation (approximately 8 million cells in each 10 cm dish) might be

Fig. 1 .
Fig. 1.Overview of mDA progenitor screening protocol.A. mDA neuronal differentiation.Basal media and patterning factors required for each stage of the process are shown.On Day 11, compounds are dispensed from the source plates onto PO/FN/LN-coated, dried, plates.Subsequently, MDa progenitors are plated and cultured for another 24 h prior to fixing and staining.B. Image Analysis.Representative images.LC3 puncta and nuclei are counted for each imaged field, and LC3 puncta / nuclei values are calculated.Through image segmentation, LC3 puncta outside the cytoplasm (background noise) are excluded from the analysis.Abbreviations: EBs: embryoid bodies, FN: fibronectin, LN: laminin, mDA: midbrain dopaminergic, PO: poly-L-ornithin.

Fig. 2 .
Fig. 2. Characterization of ventral midbrain progenitors at Day 11 of differentiation.A. Immunofluorescence.Expression of ventral midbrain markers forkhead box protein A2 (FOXA2) and homeobox transcription factor 1 alpha (LMX1A) at Day 11 of differentiation.Co-expression of FOXA2 and LMX1A is thought to be unique to the ventral midbrain, hence a high percentage of doubly-positive cells indicates effective initiation of differentiation into mDA neurons.Quantification via counting doubly-positive cells (e.g., on ImageJ) is recommended; the relevant quantification graph suggests effective differentiation into ventral midbrain progenitors as per relevant literature [ 17 , 18 ].B. Real-Time Quantitative Reverse Transcription PCR (qRT PCR).Day 11ventral midbrain progenitors tested for appropriate gene expression suggestive of effective initiation of mDA neuronal differentiation.For each marker/ target, Δ  mean values on Day 11 (calculated using the target's   mean values, normalised versus the ones from the housekeeping gene GAPDH) are compared to ones in corresponding iPSCs.The line exhibits downregulation in gene expression of pluripotency-related markers OCT4 and NANOG , as well as upregulation in gene expression of ventral midbrain markers FOXA2, LMX1A, LMX1B, EN1 and EN2 .When such immunofluorescence marker ratios and qRT PCR marker patterns are achieved, one can be confident of effective generation of ventral midbrain progenitors; if the differentiation shows different patterns, cells must be discarded, and new differentiations started.

Fig. 1 A
, Fig.3)1 Coat the 96-well low skirted microplates with PO/Fn/LN using the Beckman Coulter Biomek NXp and Biotek EL406 automated workstation and the SAMI scheduling software, as follows:

Fig. 3 .
Fig. 3. Automated compound delivery.A. Schematic drawing of the 384 LDV Echo source plate containing the first 80 individual drugs of a compound library.The library can be dispensed into 96 well assay plates using the Labcyte Echo 550 Acoustic dispenser.B. Final plate layout in the 96 well assay plate containing controls and library compounds.Table2Automated PFA fixation.The stations the assay plates run through during the fixation process are listed in the left column.The individual steps of the fixation protocol at the respective stations are described in the right column.

Fig. 4 .
Fig. 4. Assay reproducibility and capacity for high content.A .To assess reproducibility, a pilot source plate was created containing i) compounds known to enhance LC3 puncta production (either by inducing autophagy flux or blocking autophagosome to lysosome fusion), ii) compounds with no expected effect on LC3 puncta, as well as iii) negative and positive controls (DMSO and Bafilomycin A1 in columns 1 and 12, respectively).After treatment with these compounds, destination plates in technical triplicates were fixed, stained and imaged.Image analysis generated heatmaps depicting increasing LC3 puncta per nuclei numbers in darker shades of orange.B. Compound treatments reproducibly generated similar numbers of LC3 puncta per nuclei in all technical replicates.In detail, we observed standard deviation for the 3 replicates ranging from 0.028 and 0.592, with mean and median values of 0.209 and 0.157 respectively (attesting to the reproducibility of the assay).C. The assay can be used to image multiple stains simultaneously [e.g., nuclear stains (DAPI), mitochondrial markers (HSP60), autophagy (LC3) and lysosomal (LAMP1) markers] and their response to different treatments, hence providing multiple parameters that can be useful for secondary hit validation.Apart from puncta numbers, multiple other parameters can also be analysed, such as cell morphology, spot area, intensity and marker co-localization.
Additionally, the media and factors needed for each stage of differentiation are detailed in Table1, Fig.1A, and the Procedure section.

Table 1
Neuronal differentiation media constitution .Shaded rows indicate constituents removed or added on specific days during the mDA neuronal differentiation protocol.

Table 3
Automated cell staining.Stations within the imaging set up are listed on the left.The respective steps for cell permeabilization, blocking, washes and primary antibody (A) as well as secondary antibody administration (B) are listed on the right.using the washer head in 3 cycles:Aspirate at travel rate 1Dispense 100 μl of PBS at flow rate 1 Plate Washer Wash with PBS using the washer head in 3 cycles:Aspirate at travel rate 1Dispense 100 μl of PBS at flow rate 1 Plate Washer Aspirate DPBS with washer head at travel rate 1 CWDispense 40 μl of secondary antibody solution with Peri-pump dispenser at low flow rate Cytomat Hotel Incubate plate at room temperature and move plate back to plate washer within 65 min Plate Washer Wash with PBS using the washer head in 3 cycles:Aspirate at travel rate 1Dispense 100 μl of PBS at flow rate 1 Cytomat Hotel Home position.Plates will be placed here, ready for imaging 2 For cell culture, work under sterile conditions in Class 2 biological safety cabinets at all times, to avoid sample contamination.Regularly test all cells for mycoplasma contamination, e.g.via the MycoAlert TM Mycoplasma Detection Kit, and immediately isolate or discard contaminated lines.
3 Discard all iPSC lines which show a spontaneous differentiation level above 5-10% and do not use for downstream differentiations (Section C1).