Differentiating PC12 cells to evaluate neurite densities through live-cell imaging

Summary Although PC12 cells are a valuable tool in neuroscience research, previously published PC12 cell differentiation techniques fail to consider the variability in differentiation rates between different PC12 cell strains and clonal variants. Here, we present a comprehensive protocol to differentiate PC12 cells into equivalent neurite densities through live-cell imaging for morphological, immunocytochemical, and biochemical analyses. We detail steps on optimized substrate coating, plating techniques, culture media, validation steps, and quantification techniques.


SUMMARY
Although PC12 cells are a valuable tool in neuroscience research, previously published PC12 cell differentiation techniques fail to consider the variability in differentiation rates between different PC12 cell strains and clonal variants. Here, we present a comprehensive protocol to differentiate PC12 cells into equivalent neurite densities through live-cell imaging for morphological, immunocytochemical, and biochemical analyses. We detail steps on optimized substrate coating, plating techniques, culture media, validation steps, and quantification techniques.

BEFORE YOU BEGIN
This protocol provides detailed instructions to differentiate distinct PC12 cell strains and clonal variants to equivalent neurite densities through live-cell imaging, and validate the differentiation through western blot analysis and immunocytochemistry. PC12 cells are commercially available for purchase from a variety of companies including ATCC and Sigma Aldrich. This protocol utilizes a PC12 Tet-Onâ cell line from Clontech, which allows for inducible expression of any gene of interest under the control of a tetracycline-inducible promoter upon treatment with Doxycycline. Experiments included here were performed with PC12 cell clonal variants that contain inducible mutant forms of the androgen receptor 1 ; for the data shown here, the androgen receptor was not expressed. Once differentiated, these neuronal cells can be used to evaluate genes, and mutant forms of these genes, involved in neurological function and disease, and to investigate their impact on neurite outgrowth and retention. For successful implementation of this protocol, researchers should have experience with techniques for general cell culture, microscopy, basic ImageJ/Fiji analysis, western blotting, and immunocytochemistry. See Figure 1 for a schematic of the experimental workflow. We recommend establishing the timing of differentiation for various PC12 cell strains and clonal variants prior to beginning validation studies. However, simultaneous plating of cells in 96-well plates for live-cell imaging and immunofluorescence, and 6-well plates for western blot analysis of neuronal markers, can be performed if desired.
Alternatives: This protocol uses the Bioruptorâ Pico sonication device for sonication of cell lysates, but an alternative sonicator may be used.
Alternatives: This protocol uses the Bio-Rad DCä Protein Assay Kit II to measure protein concentrations in cell lysates, but an alternative may be used.
Alternatives: This protocol uses the ChemiDoc Imaging System for imaging total protein and membranes for western blot, but an alternative imaging system may be used.

STEP-BY-STEP METHOD DETAILS
Coating wells-Day -1

Timing: 16-24 h
Wells in a 96-well plate are dual coated with PDL and laminin. PDL is used to promote cell adhesion, while laminin promotes attachment and differentiation of neuronal cells.
CRITICAL: Perform all steps under sterile conditions in tissue culture hood.
CRITICAL: To avoid evaporation of medium throughout the experiment, do not use the outermost wells of the plate ( Figure 2B).
Note: For initial differentiation, we recommend plating at least 3 wells per PC12 cell clonal variant to allow for quantification of neurite density during differentiation. CRITICAL: Perform all steps under sterile conditions in tissue culture hood.
Note: Pre-warm all media to 37 C prior to use.
6. Remove 96-well plate from 4 C and incubate at 20 C-25 C in tissue culture hood for 1 h. During this 1-h incubation, PC12 cells are prepared for plating (steps 7-17). 7. Aspirate PC12 cell culture medium from PC12 cells (cultured in a T25 flask) and wash cells in 1 mL DPBS without calcium or magnesium. 8. Aspirate DPBS and add 1 mL 0.05% Trypsin EDTA.
a. Incubate flask in a 37 C cell culture incubator with 5% CO 2 for 2 min. 9. Add 1 mL PC12 cell culture medium to inactivate trypsin.
a. Triturate cells 5-10 times with a P1000 pipette tip to thoroughly separate cells, while avoiding the formation of bubbles.
Note: For this protocol, PC12 cells were cultured in a T25 flask prior to plating for differentiation. The above volumes may need to be adjusted if a larger volume flask or different culture vessel is used.
10. Pass cells through a 40 mm sterile cell strainer into a 50 mL sterile centrifuge tube. 11. Centrifuge cells for 3 min at 200 3 g at 20 C-25 C. 12. Aspirate the liquid from the tube, leaving the cell pellet undisturbed. 13. Resuspend the cell pellet in 1 mL PC12 cell culture medium and transfer the PC12 cell suspension to a 1.5 mL sterile microcentrifuge tube. 14. Pass the PC12 cell suspension through a sterile 26-gauge syringe needle 15 times to create a single cell suspension (troubleshooting 1).
Note: Work slowly to avoid forming bubbles.
15. Count cells on a hemocytometer. a. Create a 1:10 dilution of PC12 cell suspension by combining 10 mL PC12 cell suspension and 90 mL PC12 cell culture medium, and add 20 mL to the hemocytometer. 16. Calculate the volume of PC12 cells required per well to achieve a plating density of 1.0 3 10 4 cells/cm 2 . Use the following formula, where ''x'' is the volume of cell suspension per well (mL): x = À Desired plating density ðcells cm 2 Þ Á À Growth area per well ðcm 2 Þ Á

Number of cells per mL ðcells=mLÞ
a. For the 96-well plates used in this protocol, the growth area per well is 0.33 cm 2 . 17. Prepare diluted PC12 cell suspension for plating.
a. Calculate the total volume of PC12 cell culture medium needed to plate a final volume of 100 mL per well (+ 3 extra wells) and aliquot into a new sterile tube. b. Multiply the calculated volume of cells per well (step 16) by the total number of wells being plated (+ 3 extra wells) to determine the volume of PC12 cell suspension to add to the aliquoted PC12 cell culture medium. c. From the aliquot of PC12 cell culture medium, remove and discard the volume calculated in step 17b and add the calculated volume of PC12 cell suspension to make the diluted PC12 cell suspension for plating. i. Pipette up and down a few times to mix.

OPEN ACCESS
Note: Before preparing the diluted PC12 cell suspension, pipette the PC12 cell suspension up and down a few times to mix (troubleshooting 1).
18. Wash each well one at a time by aspirating the laminin and immediately adding 100 mL PC12 cell culture medium.
Note: Working one well at a time ensures that the wells do not dry out, which is crucial to prevent laminin inactivation and cell detachment during culture.
Note: If the 1-h 20 C-25 C incubation for the 96-well plate (step 6) is completed prior to completing steps 16 and 17, perform step 18 first.
19. Keeping the 96-well plate flat on the tissue culture hood surface, aspirate the medium from the wells one well at a time and add 100 mL diluted PC12 cell suspension to each well ( Figure 2C).
CRITICAL: Do not let wells dry out while plating cells. Aspirate one well at a time and immediately add the diluted PC12 cell suspension.
Note: Invert the diluted PC12 cell suspension after plating every 3-6 wells to prevent cells from settling to the bottom of the tube.
Note: Pipette diluted PC12 cell suspension directly in the center of the well ( Figure 2C) (troubleshooting 1).
20. Fill the unused surrounding wells in the 96-well plate with 200 mL DPBS.
CRITICAL: Do not tilt the cell culture plate while plating cells or adding DPBS to empty wells, as this will cause uneven distribution of cells throughout the well and may lead to cell clumping in one region ( Figure 2C) (troubleshooting 1).

21.
Carefully transfer the 96-well plate to a 37 C cell culture incubator with 5% CO 2 , avoiding tilting the plate to prevent uneven distribution of cells.
Inducing neuronal differentiation-Day 0 i. Pipette up and down a few times to mix. 24. One well at a time, remove the PC12 cell culture medium gently by pipette and discard, and add 150 mL working NGF solution.
CRITICAL: Do not let wells dry out while changing media. Change media one well at a time and immediately add working NGF solution.
Note: Gently tilt the plate and put the pipette tip at the edge of the well to remove media. Do not aspirate media to avoid cell detachment. Slowly dispense media down the wall of the well to add. Do not add media directly into the well to avoid disrupting the cells.
25. Store cells in a 37 C, 5% CO 2 cell culture incubator for the duration of cell culture.
Live-cell imaging of differentiating PC12 cells-Day 0 and daily until end of experiment

Timing: 15 min per day
Differentiating PC12 cells undergo live-cell imaging daily to quantify neurite density over time.
Note: The following steps describe live-cell imaging on an EVOS M7000 microscope. Set-up may vary with alternative microscopes.
Note: If PC12 cells are imaged for longer than 20-30-min increments, use an onstage incubator set to 37 C, 5% CO 2 . If cells are imaged for longer than 1-h increments, also include humidity (80%).
26. Immediately after NGF treatment, image a set region of each well to track differentiation in the same fields of view over time. We recommend the following parameters: a. Use a 203, short working distance objective. b. Use a transmitted light filter cube with phase contrast. c. Image 10% of each well from the center.
Note: A different imaging area can be used, as long as this area remains consistent across time points and cell strains.
Note: There is potential for a slight shift in the population of cells being captured each day due to shifts in the alignment of the plate in the vessel holder and cell migration, although this is very minimal. Capturing a set area of cells as opposed to selecting random fields of view throughout the well greatly reduces this risk and ensures the population of cells being analyzed remains consistent, since the impact of changes in cell location is minimal in a large area compared to a single field of view.
d. Use autofocus set to ''small structure'' for optimal focus on the cells and neurites (troubleshooting 2). e. Save the data as a stitched tiled imaged to use for quantification of neurite density. 27. Repeat step 26 every 24 h to acquire images of the same fields of view to quantify neurite density over time.
Note: Once the total neurite density reaches $1,500 mm/mm 2 in a 0.033 cm 2 area (the total neurite length in 10% of one well), the cells are at a sufficient level of differentiation for secondary analyses (see quantifying neurite density over time). For the clonal variants used in this protocol, the desired total neurite density was reached in 3-6 days (Figure 3), although this window may be shorter or longer for other PC12 cell strains or clonal variants.

OPEN ACCESS
Note: Neurites can continue to grow longer, and undifferentiated cells can grow new neurites, after the total neurite density in the well reaches $1,500 mm/mm 2 . Cells can be differentiated past this neurite density and used for secondary analyses, but Cytosine-beta-D-arabinofuranose hydrochloride (AraC) treatment may be needed to inhibit proliferation of undifferentiated cells and limit cell clumping (troubleshooting 3) (Figure 4). AraC interferes with DNA synthesis and therefore results in cell death of dividing cells without affecting neuronal cell survival or neurite outgrowth. 4 Maintaining differentiating PC12 cell culture-Day 2 and every 2 days until end of experiment

Timing: 1 h every 2 days
Differentiating PC12 cells are supplemented with NGF, CultureOne, and GlutaMAX every 2 days through half-feed media changes until PC12 cells reach a total neurite density of $1,500 mm/mm 2 . CultureOne is added to the medium to inhibit proliferation of undifferentiated cells during the differentiation process. AraC can also be used if there is an abundance of proliferation and/or cell clumping (troubleshooting 3) (Figure 4).
CRITICAL: Perform all steps under sterile conditions in tissue culture hood.

OPEN ACCESS
30. Remove and discard 75 mL of media from each well and add 75 mL stock differentiation medium supplemented with NGF, CultureOne, and GlutaMAX. 31. Repeat steps 28-30 every two days until the neurite density reaches $1,500 mm/mm 2 (see quantifying neurite density over time).

Validating neuronal differentiation-Day 0 through end of validation
Timing: 4-6 days, in addition to cell culture time Differentiated PC12 cells should express the neuronal proteins b-III-Tubulin, Synapsin-I, and GAP43. 3,5,6 To verify expression of these proteins, differentiating PC12 cells are lysed throughout the differentiation process and neuronal proteins are detected through western blot analysis. Once PC12 cells have differentiated to a total neurite density of $1,500 mm/mm 2 , cells are fixed and evaluated for expression of the neuronal markers through immunocytochemistry.

Western blot analysis
Timing: 2-3 days, in addition to cell culture time PC12 cells are plated on PDL-coated 6-well plates for differentiation. Cell lysates are collected for western blot analysis of neuronal proteins on Day 0 and every subsequent 2 days until the total neurite density reaches $1,500 mm/mm 2 (as determined through steps 26 and 27 and quantifying neurite density over time).
Note: Depending on the differentiation rate of the cells, lysates may need to be collected every day to observe the increase in neuronal proteins over time.

Coating wells
Timing: 1 h 32. Sterilize wells in 6-well plates under UV light in tissue culture hood for 30 min, with lids removed from the plates. a. Cells will be plated in triplicate for each timepoint at which cell lysates are collected. For example, if collecting lysates on days 0, 2, 4, and 6, a total of 12 wells will be required for the experiment. b. Cells to be lysed on different days need to be plated in separate 6-well plates. Therefore, the above example requires 4 plates. c. Label each plate with the day that cells will be lysed (i.e., ''Day 0'', ''Day 2'', ''Day 4'', or ''Day 6''). 33. Prepare a 20 mg/mL working laminin solution by diluting laminin stock solution in cell culture grade water. a. Add 1 mL working laminin solution to each well. b. Wrap plates in parafilm and incubate for 16-24 h at 4 C.
Note: Although not tested in this protocol, alternative 6-well plates that are not pre-coated with PDL might be suitable for differentiation, but wells must be coated with PDL (1.5 mL 50 mg/mL PDL per well) prior to laminin coating. 34. Follow steps 6-21 to plate PC12 cells, with the following alterations: a. In step 16, plate PC12 cells at a density of 1.5 3 10 4 cells/cm 2 . This will yield a high enough protein concentration in the cell lysates to detect neuronal proteins through western blot. b. In step 17, a final volume of 2 mL will be added to each well of a 6-well plate. c. In step 18, add 1 mL PC12 cell culture medium to each well. d. In step 19, add 2 mL diluted PC12 cell suspension to each well. e. In step 20, fill the unused surrounding wells with 2 mL DPBS.

Inducing neuronal differentiation
Timing: 1 h 35. Follow steps 22-25 to induce neuronal differentiation for all plates except ''Day 0'' plate, with the following alterations: a. In step 22, prepare at least 50 mL stock differentiation medium (this volume will vary based on the number of wells being plated, so calculate the exact volume needed for the duration of the experiment before preparing media). b. In steps 23 and 24, a final volume of 2 mL will be added to each well. CRITICAL: PMSF is toxic if swallowed and can cause skin corrosion. Wear proper PPE when handling.

Lysing cells, sonicating cell lysates, and measuring protein concentration
Note: Add PMSF to lysis buffer immediately before lysing cells.
Note: Although not tested in this protocol, an alternative lysis buffer may be used for cell lysis.
b. Aspirate media from the wells and wash cells in 1 mL DPBS with calcium and magnesium. c. Aspirate DPBS and add 30 mL working Triton DOC lysis buffer to each well.
Note: Make sure all DPBS is aspirated from the well before adding lysis buffer to avoid diluting the cell lysates (troubleshooting 4).
d. Scrape the cells in the well thoroughly in multiple directions with a mini cell scraper and add the cell lysate to a tube for sonication. e. Centrifuge lysates for 2-5 s in a mini centrifuge. 37. Sonicate cell lysates using a sonicator. In this protocol, the Bioruptorâ Pico sonication device was set to 4 C and sonication settings were 6 cycles, 30 s on, 30 s off.
Pause point: Lysates can be stored at À80 C.
38. Measure protein concentrations in cell lysates using the Bio-Rad DC Protein Assay Kit II or other suitable alternative, according to standard protocol. 39. Store lysates at À80 C until all cells are lysed on subsequent days before running western blot.
Optional: Lysates can be aliquoted prior to storing at À80 C to avoid excess freeze/thaw cycles if multiple western blots need to be run. Make aliquots in triplicate of at least 15 mg protein for each sample.
40. Repeat steps 36-39 every 2 days to lyse cells at multiple time points during the differentiation process.
Note: The volume of lysis buffer added to each well will likely need to be increased for lysis of cells on later days of the differentiation process to account for the increase in cell density/neurite outgrowth over time. See Figure 5 for details on cell confluency and corresponding lysis buffer volume and protein concentration (troubleshooting 4).

Maintaining differentiating PC12 cell culture
Timing: 1 h every 2 days 41. Follow steps 28-30 every 2 days to maintain the culture of differentiating PC12 cells through half-feed media changes with the following alterations: a. In step 28, a final volume of 1 mL per well is needed. b. In step 30, remove and discard 1 mL of media from each well and add 1 mL stock differentiation medium supplemented with NGF, CultureOne, and GlutaMAX.
Note: Cells to be lysed on a specific day do not need to undergo a half-feed media change on that day. Therefore, every subsequent 2 days of the protocol, one fewer plate of cells will undergo a half-feed media change. Recalculate the volumes of media, NGF, CultureOne, and GlutaMAX needed for cell culture every 2 days to avoid wasting reagents. c. Prepare 13 running buffer from 103 running buffer stock solution. Add 0.1% SDS to the 13 running buffer. d. If not already done, aliquot at least 15 mg of protein from each cell lysate sample, preparing triplicate aliquots of each sample in order to run 3 gels. i. To these aliquots, add an appropriate volume of 23 sample buffer + 2-Mercaptoethanol to the lysates for a final concentration of 13 and vortex each sample. e. Boil samples at 90 C-100 C for 5 min. f. Vortex samples and centrifuge for 5 s in a mini centrifuge. g. Load identical samples into 3 SDS-PAGE stain-free gels and run at 120 V in 13 running buffer + SDS.
Note: Running three separate gels with identical samples allows detection of the three differentiation markers Synapsin-1, b-III-Tubulin, and GAP43 at the same time. Alternatively, two gels can be run, and after protein transfer and immunoblotting, one membrane can be stripped of bound antibody and reprobed for the third protein.
Optional: If desired, PC12 (NGF-differentiated) lysates (ECM Biosciences Cat#PL7141) can be loaded on the gel as a positive control.
Note: This protocol uses stain-free gels (poured using TGX Stain-Freeä FastCastä Acrylamide Kit, 10%) for SDS-PAGE to normalize neuronal proteins of interest to total protein in each well. We have not evaluated whether expression of housekeeping genes used for normalization change with differentiation and therefore cannot guarantee their accuracy as a measure of total protein.
44. After the dye front has left the gels, image gels for total protein using a ChemiDoc or other compatible imaging system. 45. Transfer proteins from the gels to 0.45 mm polyvinylidene difluoride (PVDF) membranes in 13 transfer buffer according to standard protocol. 46. Incubate the membranes with the following blocking solutions for 1 h at 20 C-25 C: ll OPEN ACCESS a. Membranes 1 and 2: 5% nonfat dry milk in 1 3 0.05% TBST (these membranes will be probed for Synapsin-1 and GAP43). b. Membrane 3: 5% BSA in 1 3 0.05% TBST (this membrane will be probed for b-III-Tubulin). 52. Develop membranes with Clarity Western ECL Substrate using a ChemiDoc or other compatible imaging system. 53. Quantify total protein lanes and protein bands using Image Lab software or other quantification software. Normalize protein bands to total protein for each well.

Immunocytochemistry
Timing: 2-3 days Following differentiation of PC12 cells to a neurite density of $1,500 mm/mm 2 (as determined in steps 26 and 27 and quantifying neurite density over time), cells are fixed and immunostained for Synapsin-1, GAP43, and b-III-Tubulin directly in the 96-well plate used for differentiation.
CRITICAL: All liquid should be removed from the wells gently by pipette to avoid loss of cells and neurites during the staining process (troubleshooting 5). Remove and add liquid 3 wells at a time to prevent cells from drying out.
CRITICAL: Secondary antibodies and Hoechst dye are light-sensitive. Protect the plate from light once they are in use.
Note: Immunocytochemistry is performed after completing steps 1-31. These steps do not need to be performed under sterile conditions.
Note: Although not tested in this protocol, cells may be plated on coverslips for differentiation and subsequent immunocytochemistry. a. Remove and discard DPBS wash from the wells and add 100 mL NGS/Triton X-100 solution per well. b. Incubate for 1 h at 20 C-25 C. 56. Prepare primary antibody solutions in DPBS with calcium and magnesium + 5% NGS.

Fix cells in paraformaldehyde (PFA
a. Half of the wells will be incubated with b-III-Tubulin (Ms, 1:1000) and Synapsin-1 (Rb, 1:1000) primary antibodies. b. The other half of the wells will be incubated with b-III-Tubulin (Ms, 1:1000) and GAP43 (Rb, 1:100) primary antibodies. c. Remove and discard the permeabilization/blocking solution and add 80 mL of the appropriate primary antibody solution to each well. d. Wrap the plate in parafilm and incubate for 16-24 h at 4 C. 57. Wash cells 3 times for 10 min in 100 mL DPBS with calcium and magnesium.
a. Put the plate on an orbital shaker at the lowest speed during washes (troubleshooting 5). 58. Prepare secondary antibody solution in DPBS with calcium and magnesium + 5% NGS.
a. All wells will be incubated with Goat anti-Mouse IgG Highly Cross-Adsorbed, Alexa Fluor 594 (1:500-1:1000) and Goat anti-Rabbit IgG Highly Cross-Adsorbed, Alexa Fluor 488 (1:500) secondary antibodies. b. Remove and discard the DPBS wash from the wells and add 80 mL secondary antibody solution to each well. c. Wrap the plate in aluminum foil to protect from light and incubate for 1 h at 20 C-25 C. 59. Wash cells 3 times for 10 min in 100 mL DPBS with calcium and magnesium.
a. Wrap the plate in aluminum foil to protect from light and incubate on an orbital shaker at the lowest speed during washes (troubleshooting 5). 60. To stain nuclei, prepare 1:500 Hoechst dye solution in DPBS with calcium and magnesium + 5% NGS from a 1 mg/mL working stock solution. a. Remove and discard DPBS wash from the wells and add 80 mL Hoechst solution to each well. b. Wrap the plate in aluminum foil to protect from light and incubate for 20 min at 20 C-25 C. 61. Remove and discard the Hoechst solution and add 100 mL DPBS with calcium and magnesium to each well.
Pause point: Before imaging, the plate can be wrapped in aluminum foil and parafilm and stored at 4 C for up to 2 weeks. 62. Image neurite-bearing cells with a fluorescence microscope equipped with a 96-well plate holder. We recommend using a 203 or 403 short working distance objective.

EXPECTED OUTCOMES
Using this protocol, researchers will be able to differentiate PC12 cells into neurite-bearing cells that express the neuronal markers Synapsin-1, GAP43, and b-III-Tubulin. These cells can be used to evaluate the role of proteins in neurological function and disease, and to investigate their impact on neurite outgrowth and retention. As PC12 cells differentiate, they undergo a change in cell body shape from circular to triangular and gradually extend neurites that develop bulbous terminal ends (Figure 6, arrows). This protocol details how to differentiate varied PC12 cell strains and clonal variants to equivalent stages of differentiation through live-cell imaging, and quantify neurite density over time. We show that 4 different PC12 cell clonal variants, each containing a different inducible mutant form of the androgen receptor (not expressed in these experiments), required between 3-6 days of differentiation before reaching the target neurite density of $1,500 mm/mm 2 , representative of the variability in differentiation rates between clonal variants. These different clonal variants also varied in the propensity of cells to clump together and proliferate, which may also impact time to differentiate (Figure 3).
Expression of Synapsin-1, GAP43, and b-III-Tubulin should increase over time of differentiation. 3,5,6 We show that these neuronal proteins are absent or expressed at very low levels in undifferentiated PC12  Live-cell images acquired daily are analyzed in Fiji 2 to quantify the total neurite density in the same fields of view over time. It is best to analyze images on the day they are captured to determine whether continued differentiation is required to reach the target neurite density of $1,500 mm/mm 2 .
1. Starting with images acquired on day 1 of differentiation, open a tiled image file in Fiji ( Figure 8A).

LIMITATIONS
This protocol does not yield 100% differentiation of PC12 cells. The percent of cells that are differentiated varies between clonal variants and may be impacted by the distribution of cells in the well while plating or the propensity of cells to clump and proliferate. Performing single cell analyses such as immunocytochemistry avoids this limitation by allowing the researcher to select the differentiated cells for analysis. However, bulk analyses such as western blot cannot exclude undifferentiated cells and are therefore not completely accurate measures of the differentiated population. To analyze differentiated cells through western blot, normalize the protein(s) of interest to b-III-Tubulin expression for each sample, which will account for differences in the percent of cells that are differentiated between cell lines/wells.

Potential solution
Cells might be clumped together in the well due to failure to create a single cell suspension before plating or poor plating technique.
Creating a single cell suspension: To create a single cell suspension, it is crucial to follow steps 7-14 exactly, especially passing cells through a 26-gauge syringe needle. Verify that cells are not in clumps when counting on the hemocytometer (step 15). To prevent cells from re-pelleting and clumping throughout the plating steps, periodically pipette the PC12 cell suspension and diluted PC12 cell suspension up and down. Poor plating technique: Hold the pipette completely vertical and aim for the center of the well when plating cells to allow an even distribution of cells throughout the well. Keep the plate flat while plating and do not lift or tilt the plate for 5-10 min after plating to allow cells to settle in the well (generally, enough time has elapsed after filling surrounding wells with DPBS) ( Figure 2C). Hold the plate horizontal and do not tilt it when putting it in the incubator. Do not look at the cells under the microscope until 2 h have elapsed to prevent disrupting the cells before attachment to the wells.

Potential solution
Autofocus can fail if cells are sparsely plated, as needed in this differentiation protocol. Changing the field acquisition order so that a different field of view is imaged first may resolve the issue, but it is not guaranteed. If autofocus continues to fail, manually focus each well prior to imaging.

Problem 3
Cells begin to clump and/or the culture is overtaken by proliferation of undifferentiated cells when differentiating past a neurite density of $1,500 mm/mm 2 (step 27).

Potential solution
Treat cells with cytosine-beta-D-arabinofuranose hydrochloride (AraC) for 48 h at a final concentration of 1 mM starting on the day cells reach the target neurite density. AraC interferes with DNA synthesis and therefore results in cell death of dividing cells without affecting neuronal cell survival or neurite outgrowth. 4 We have verified that AraC treatment eliminates proliferating cells and reduces clumping of neurite-bearing cells with undifferentiated cells (Figure 4).
Note: The plating density may need to be increased to 1.5 3 10 4 or 2.0 3 10 4 cells/cm 2 to account for the decrease in cell number from AraC-induced cell death.

Problem 4
Low protein concentration in cell lysates (western blot analysis steps 36 and 40).

Potential solution
Completely aspirate all DPBS prior to adding lysis buffer to the wells to avoid diluting the cell lysates. It may also be necessary to decrease the volume of lysis buffer added to each well. Use Figure 5 as a guide to estimate starting lysis buffer volumes based on cell confluency and adjust as necessary.

Problem 5
Abundance of neurite/cell loss after performing immunocytochemistry (immunocytochemistry steps 57 and 59). Protocol Potential solution Cell loss during the staining process may be due to improper coating of the wells (see coating wells). Verify that PDL is the proper molecular weight of 50,000-150,000 Daltons (best for use in neuronal culture), laminin aliquots are less than 6 months old, and laminin is thawed and used on ice. During the staining process, remove all liquid gently by pipette by placing the pipette against the edge of the well. Be very gentle when dispelling liquid down the side of the well. Washes may be performed without the use of the orbital shaker to prevent potential cell detachment, but this may result in slightly increased background signals.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact [Diane E. Merry] (diane.merry@jefferson.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate any datasets or code.