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Method Article

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

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DOI:

10.3791/60440

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November 12th, 2019

 ,  ,  ,  , 
1Department of Neurology, Boston Children's Hospital, 2FM Kirby Neurobiology Center, Boston Children's Hospital, 3Department of Neurology, Harvard Medical School, 4Medical Genetics Training Program, Harvard Medical School, 5Department of Ophthalmology, Boston Children's Hospital, 6Department of Ophthalmology, Harvard Medical School, 7Broad Institute of M.I.T. and Harvard, 8Howard Hughes Medical Institute, 9Department of Neurology, Kokura Memorial Hospital, 10Department of Genetics, Albert Einstein College of Medicine

In This Article

Summary

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Abstract

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Introduction

The culture of primary motor neurons is a powerful tool which enables the study of neuronal development, function, and susceptibility to exogenous stressors. Motor neuron cultures are particularly useful for the study of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS)1,2, whose disease mechanisms are incompletely understood. Interestingly, despite the significant cell death of spinal motor neurons (SMNs) in both ALS patients and ALS model mice, cell death in oculomotor neurons (CN3s) and trochlear neurons (CN4s) are relatively scarce1,3,4,5,6,7,8,9. Therefore, comparative analyses of pure cultures of CN3s/CN4s and SMNs could provide important clues about mechanisms underlying relative vulnerability. Unfortunately, a major barrier to such analyses has been the inability to grow purified cultures of these motor neurons.

Many protocols have been described for the purification of SMNs from animal models. Most of these protocols use density gradient centrifugation10,11,12 and/or p75NTR-antibody-based cell-sorting panning techniques13,14,15,16. Density gradient centrifugation exploits the larger size of SMNs relative to other spinal cells, whereas p75NTR is an extracellular protein expressed exclusively by SMNs in the spinal cord. Nearly 100% pure SMN cultures have been generated by one or both of these protocols11,12,14. However, these protocols have not been successful in generating CN3/CN4 cultures because CN3s/CN4s do not express p75NTR, and other specific CN3/CN4 markers have not been identified. They are also smaller than SMNs and, therefore, more difficult to isolate based on size. Instead, in vitro studies of CN3s or CN4s have relied on dissociated17,18,19,20,21, explant17,22,23,24,25,26, and slice27,28 cultures, which are composed of heterogeneous cell types, and no protocols have existed for the isolation and culture of primary CN3s or CN4s.

Here, a protocol is described for the visualization, isolation, purification, and cultivation of CN3s, CN4s, and SMNs from the same embryonic day 11.5 (E11.5) IslMN:GFP transgenic mice29 (Figure 1, Figure 2A). IslMN:GFP specifically labels motor neurons with a farnesylated GFP that localizes to the cell membrane. This protocol enables species- and age-matched comparison of multiple types of motor neurons in order to elucidate pathological mechanisms in motor neuron disease.

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Protocol

All experiments utilizing laboratory animals were performed in accordance with NIH guidelines for the care and use of laboratory animals and with the approval of the Animal Care and Use Committee of Boston Children's Hospital.

1. Setting Up Timed Matings Prior to the Dissection

  1. To generate prenatal embryonic mice for motor neuron harvest, weigh each female mouse and set up timed mating between adult IslMN:GFP transgenic mice 11.5 days prior to the day of neuron isolation. For the purpose of developing this protocol, 129S1/C57BL/6J IslMN:GFP mice, aged 2−9 months, were used and timed mating was set up in the evening.
  2. Examine female mice for vaginal plugs the following morning. Consider the date on which the plug is identified as embryonic day (E) 0.5.
  3. Weigh female mice and examine for pups using ultrasound (see Table of Materials) between E8.5−11. Check for the signs of successful mating.
    1. Confirm the successful mating by detecting weight gain in female mice (usually >1.5 g on E9.5 if there are more than 5−6 embryos).
    2. Visually confirm embryos under ultrasound. Embryos are easily detectable by ultrasound after E9.5. Ultrasounds are conducted only on females that have gained weight because they are more often pregnant than those that do not.
      NOTE: Female mice can gain weight for reasons other than pregnancy, so weight gain alone is not a reliable indicator of pregnancy. Ultrasound confirmation prevents unnecessary sacrifice of females that are not pregnant but is not crucial if unavailable.

2. Dissection Conditions and Preparation of Instruments

  1. Perform all coating (except acid-cleaning of coverslips), media preparation, tissue dissociation (except centrifugation and incubation), and culture work in a laminar flow hood to ensure the sterility of the media and embryonic motor neurons.
  2. With careful attention to sterile technique, conduct tissue dissection outside of a laminar flow hood with minimal risk of contamination.
  3. Sterilize one dissection plate, one pair of microdissecting scissors, one pair of thumb dressing forceps, two pairs of Dumont #5 tweezers, one microdissecting knife, and one Moria mini perforated spoon by immersing in 70% ethanol prior to use.

3. PDL/Laminin Coating of Dishes/Coverslips

NOTE: Culture dissociated primary motor neurons in 96 well or 24 well plates, depending on the number of cells required for the application. Cells can be imaged directly in the tissue culture plate without the use of coverslips if the wells are optically transparent and the thicknesses are compatible with imaging.

  1. For applications requiring coverslips, prepare acid-cleaned, sterilized, and air-dried coverslips at least 2 days prior to the neuron isolation as previously described30. Batches of coverslips can be prepared in this manner well in advance of experiments and can be stored up to 6 months without impact on experimental quality.
  2. Prepare a working solution of 20 µg/mL poly D-lysine (PDL) in phosphate buffered saline (PBS) 2 days prior to the neuron isolation.
    1. Aliquot PDL (1 mg/mL) in advance and store at -20 °C as a stock solution.
  3. Cover the surface of each coverslip or the well of the tissue culture plate with enough PDL solution working solution (e.g., 100 µL per well on 96 well plates or 500 µL per well on 24 well plates with or without coverslips). Incubate overnight at 37 °C.
  4. The following day wash 3x with sterilized water.
    1. Seal the plates with paraffin film and store the washed PDL-coated plates at 4 °C for up to 1 month if they are dried completely after the final wash.
  5. Prepare a working solution with 10 µL of laminin (1.1−1.2 mg/mL) in 1.2 mL of PBS.
    1. Aliquot laminin stocks (1.1−1.2 mg/mL) in advance and store at -80 °C.
  6. Cover the surface of each coverslip or well with enough laminin solution to evenly coat the surface. Incubate for at least 2 h at 37 °C prior to use.
    NOTE: PDL/laminin-coated plates and coverslips can be stored up to 1 week at 37 °C, but freshly coated laminin is preferred. Remove laminin directly before plating30. Laminin should not be allowed to dry out. If the plates are to be stored for more than several hours, they should be wrapped in paraffin film.

4. Preparation of Dissection, Motor Neuron Culture Media, and Dissociation Solutions

NOTE: Concentrations in parentheses indicate the final concentrations of each reagent.

  1. Prepare the dissection medium.
    1. Thaw the heat-inactivated horse serum overnight at 4 °C, prepare 1 mL aliquots, and store at -20 °C. Thaw aliquots at RT directly before use.
    2. Store B27-supplement (50x) in 1 mL aliquots at -20 °C. Thaw aliquots at RT immediately before use. Avoid freeze/thaw cycles.
    3. Store glutamine supplement (100x) at 4 °C or -20 °C. Divide into 0.5 mL aliquots if storing at -20 °C.
    4. Store penicillin-streptomycin (10,000 U/mL) in 1 mL aliquots at -20 °C. Thaw aliquots at RT immediately before use.
    5. To make the dissection medium, mix 9.4 mL of Hibernate E with 200 µL of horse serum (2%), 200 µL of 50x B27 supplement (1x), 100 µL of 100x glutamine supplement (1x) (e.g., GlutaMAX), and 100 µL of 10,000 U/mL penicillin-streptomycin (100 U/mL). Use this medium for collecting dissected tissues and making the final suspension of dissociated cells.
    6. Add 500 µL of the dissection medium to individual 1.7 mL microcentrifuge tubes for the tissue collection the day before the neuron isolation. Prepare one tube for each tissue type that will be collected (e.g., positive control, negative control, CN3/CN4, SMN) and store at 4 °C prior to the use.
    7. Combine 49 mL of Hibernate E low fluorescence media with 1 mL of B27 supplement (1x) and fill a 24 well plate with this medium (2 mL/well) the day before the neuron isolation. Use this dish for collecting mouse embryos. Store at 4 °C prior to use.
  2. Prepare the motor neuron culture medium.
    1. Prepare 250 µL aliquots of 25 mM 2-mercaptoethanol in Leibovitz's L15 medium. Store at -20 °C.
    2. Generate 5 µL aliquots of 100 µg/mL solutions of BDNF, CNTF, and GDNF diluted in sterilized water. Store at -80 °C and thaw aliquots at RT immediately before use.
    3. Prepare 10 mM forskolin solution by adding 64 µL of dimethyl sulfoxide (DMSO) to 5 mg (1.0670 µM) of forskolin and vortex well to dissolve completely. Then add sterilized water (1.003 mL) to the DMSO solution and vortex well. Store 12 µL aliquots of 10 mM forskolin at -20 °C and thaw aliquots at RT immediately before use.
    4. Prepare 12 µL aliquots of 100 mM isobutylmethylxanthine (IBMX) diluted in DMSO. Store at -20 °C and thaw aliquots at RT immediately before use.
    5. To make the motor neuron culture medium, mix 9.4 mL of neurobasal medium with 200 µL of horse serum (2%), 200 µL of 50x B27 supplement (1x), 100 µL of 100x glutamine supplement (1x), 100 µL of 10,000 U/m: penicillin-streptomycin (100 U/mL), and 20 µL of 25 mM 2-mercaptoethanol (50 µM), preferably directly before use. This step can be performed up to 1 day before neuron isolation.
    6. Just before the use, add 1 µL each of 100 µg/mL BDNF (10 ng/mL), CNTF (10 ng/mL), and GDNF (10 ng/mL) and 10 µL of 10 mM forskolin (10 µM), and 10 µL of 100 mM IBMX (100 µM) to the motor neuron culture medium. Prewarm the medium to 37 °C.
  3. Prepare the dissociation solutions.
    1. Prepare the papain solution (20 U/mL papain and 0.005% DNase) and an albumin-ovomucoid inhibitor solution (1 mg/mL ovomucoid inhibitor, 1mg/mL albumin, and 0.005% DNase) following the manufacturer's instructions.
    2. Prepare 500 µL aliquots of each of ovomucoid inhibitor and papain solutions and store at -80 °C. Thaw aliquots at 37 °C immediately before use.

5. Ventral midbrain and Spinal Cord Dissection

NOTE: Perform all of the following steps except for steps 5.1.1−5.1.3 and 5.1.5−5.1.6 under a fluorescence dissection stereomicroscope. Total dissection time per experiment is typically 3−5 h, depending on the proficiency at the dissection technique and the number of motor neurons required for each experiment.

  1. Ventral midbrain dissection
    1. Euthanize a pregnant mouse approximately 11.5 days postfertilization by carbon dioxide gas and cervical dislocation.
    2. Spray the abdomen thoroughly with ethanol and remove the uterus using sterile microdissecting scissors and thumb dressing forceps. Wash the uterus briefly in sterile PBS, then transfer to the dissection plate filled with prechilled sterile PBS.
    3. Remove the IslMN:GFP-positive embryos carefully from the uterus using sterile microdissecting scissors, thumb dressing forceps, and Dumont #5 tweezers in ice-cold sterile PBS under the bright light of the microscope. Using a sterile Moria mini-perforated spoon, transfer each embryo to a separate well of a 24 well plate filled with prechilled Hibernate-E low fluorescence medium supplemented with 1x B27. Keep the 24 well plate on ice.
    4. Transfer one embryo to a sterile dissection plate and cover it completely with ice-cold sterile Hank's balanced salt solution (HBSS).
    5. Ensure that the dissection steps are performed under fluorescein isothiocyanate (FITC) illumination of the microscope. Using tweezers, remove the tail and the face of the embryo without damaging the midbrain (Figure 2Ba). Place the embryo prone with limbs straddled underneath and tail pointing toward the front of the microscope, toward the dissector (indicated by an asterisk, Figure 2Bb).
    6. Using tweezers, slit open the roof of the fourth ventricle in order to generate a small opening. Use this opening to hook tweezers into the space created between the fourth ventricle and its roof. Dissect along the dorsal surface of the embryo rostral to the cortex and lateral to the floor plate and motor column (Figure 2Ca,b). Open the dissected tissue in an open-book manner to reveal the GFP-positive CN3 and CN4 nuclei.
      NOTE: A small piece of tissue from the ventral midbrain containing mesenchyme, CN3, and CN4 will now be exposed.
    7. Carefully separate the ventral midbrain from the embryo and remove meningeal tissue using tweezers and a microdissecting knife. Dissect the bilateral GFP-positive CN3 and CN4 nuclei away from the floor plate and other GFP-negative surrounding tissue using tweezers and a microdissecting knife (Figure 2D). Maximize the number of GFP-positive motor neurons in the excised tissue but avoid touching or damaging them.
    8. If a collection of separate CN3 and CN4 nuclei is desired, cut along the midline of these two nuclei (yellow dotted line in Figure 2D). Using a P1000 pipette, collect the dissected ventral midbrain tissue with minimal HBSS and place it in a labeled 1.7 mL microcentrifuge tube filled with dissection medium (see step 4.1.5). Store on ice until dissociation.
    9. Continue pooling ventral midbrains from additional embryos in the same tube until the total number meets the experimental requirement (refer to step 8 for ideal cell numbers).
      NOTE: A pooled collection of at least 10 ventral midbrains yielding approximately 1 x 104 CN3/CN4 motor neurons is recommended because tissues are subject to stress during the dissociation and sorting.
  2. Ventral spinal cord dissection
    1. Keep the embryo prone with the head facing the front of the microscope, toward the dissector. Hold the embryo with one pair of tweezers and insert the tip of the other pair of tweezers into the unopened caudal part of the fourth ventricle.
    2. Open the rest of the hindbrain and spinal cord dorsally over the whole rostrocaudal extent of the embryo. Open by cutting dorsal tissue, starting from the fourth ventricle and working toward the central canal of the caudal spinal cord using the forceps as scissors (Figure 2Ca,b). Take care to avoid touching or damaging the ventral spinal cord during this procedure.
    3. Hold the embryo with one pair of tweezers and pinch off the flap of the dorsal tissue on each side with the other pair of tweezers (Figure 2Ea,b).
      NOTE: Excised dorsal tissues contain dorsal skin, mesenchyme, dorsal root ganglia (DRGs), dorsal hindbrain, and spinal cord. Remove as much of these tissues as possible without damaging the SMN nuclei, because they are adhesive and can trap SMNs during filtering or cause clogging during FACS sorting.
    4. Remove the ventral spinal cord using the microdissection knife to pierce directly below the GFP-positive SMN. Lift the ventral spinal cord with saw-like movements on both sides (Figure 2Fa,b). Cut the floating ventral spinal cord transversely directly above C1, where the first GFP-positive anterior horn projects (Figure 2G). Also, cut transversely at the upper boundary of the lower limb (Figure 2G). Remove the cervical (C1)-lumbar (L2-L3) portion of the ventral spinal cord after this procedure.
    5. Place the ventral spinal cord dorsal side up and hold by pressing the GFP-negative tissue between the GFP-positive SMN columns with one pair of tweezers. Remove the remaining attached mesenchyme, DRGs, and dorsal spinal cord by trimming both sides of the GFP-positive SMN column with the microdissection knife (Figure 2H). Take care to maximize GFP-positive motor neurons without damaging them.
    6. Using a P1000 pipette, collect the dissected ventral spinal cord tissue with minimal HBSS and place in the SMN-labeled 1.7 mL microcentrifuge tube filled with dissection medium. Store on ice until dissociation. Continue pooling ventral spinal cords from additional embryos in the same tube until the total number meets the experimental requirements.
      NOTE: Collecting at least three ventral spinal cords yielding approximately 2.1 x 104 SMN is recommended because tissues are subject to stress during dissociation and sorting.
    7. Collect facial motor neurons and extremities of the IslMN:GFP mouse embryos as GFP-positive and GFP-negative controls for fluorescence-activated cell sorting (FACS), respectively. Extremities are GFP-negative because the GFP-positive axons of the SMNs have not yet extended into the extremities at this embryonic age.

6. Tissue Dissociation

NOTE: Total dissociation time is typically 1.5 h per experiment.

  1. Warm papain and albumin-ovomucoid inhibitor solution aliquots to 37 °C 30 min prior to dissociation.
  2. Briefly spin down microdissected tissues at a low speed.
  3. Using a P100 pipette, carefully remove as much Hibernate E as possible without aspirating tissues.
    NOTE: Be sure to remove all residual Hibernate E after this step to avoid reducing the efficacy of papain dissociation in the next step.
  4. Add the appropriate volume of papain solution (Table 1) to each of the 1.7 mL microcentrifuge tubes containing the microdissected tissue samples.
    NOTE: The appropriate volume of papain for the dissociation was determined in order to maximize the effective dissociation while minimizing stress on the cells.
  5. Gently triturate 8x with a P200 pipette. Perform all trituration steps gently to preserve motor neuron viability.
  6. Incubate the tubes containing the tissues for 30 min at 37 °C, agitating by finger flicking 10x every 10 min. Gently triturate each suspension 8x with a P200 pipette after incubation. Spin down the cells at 300 x g for 5 min.
  7. To ensure the efficacy of ovomucoid inhibition in the next step, use a P1000 pipette to remove and discard as much supernatant as possible without aspirating the tissues.
  8. Resuspend pellets in the appropriate volume of albumin-ovomucoid inhibitor solution (Table 1) by gently triturating 8x with a P200 pipette.
  9. Wait for 2 min to allow any remaining pieces of undissociated tissue to settle to the bottom of the tube.
  10. Collect as much supernatant as possible without aspirating undissociated tissues using a P200 pipette. Transfer the supernatant to fresh 1.7 mL microcentrifuge tubes.
  11. If some chunks of tissue remain undissociated after step 6.10, repeat steps 6.8−6.10 for the undissociated tissues that remain in the original 1.7 mL microcentrifuge tubes to maximize the final yield of dissociated cells while minimizing stress on the cells dissociated previously, contained in the supernatant of step 6.10.
  12. Spin down the cells at 300 x g for 5 min. Carefully remove and discard the supernatant using a P1000 pipette.
  13. Resuspend the pellet in the appropriate volume of dissection medium (Table 1) by pipetting 8x using a P1000 pipette. The appropriate volume of final suspension was determined so that cell density does not exceed 107 cells/mL, which can block the stream of the flow cytometry machine, but also so that cells are not excessively diluted, which results in a slowed sorting speed.
  14. Filter the suspensions through 70 µm cell strainers to eliminate any large clumps or undigested tissue. Transfer the suspensions into 5 mL round bottom polystyrene test tubes and store on ice until required.

7. Fluorescence-activated Cell Sorting (FACS)

NOTE: This protocol was optimized using a FACS sorter equipped with a 15 mw 405 nm violet laser, a 100 mw 488 nm blue laser, a 75 mw 594 nm orange laser, and a 40 mw 640 nm red laser. Cells were sorted as sheath fluid in sterile PBS under aseptic conditions through a 100 µm nozzle. In order to minimize cell stress, the flow rate was set to a sample pressure of 1−3, such that a maximum of 1,000−4,000 events per second were acquired. Total FACS time is typically 1−2 h per experiment.

  1. Set up voltages for forward and side scatter so that the cell population can be visualized properly. Setting up appropriate voltages for cell sorting is complex and requires an experienced FACS operator.
  2. To distinguish different cell populations, plot cells based on size as determined by the Forward Scatter Area (FSC-A) versus internal complexity as determined by the Side Scatter Area (SSC-A). Draw a gate around the live cells as indicated in Figure 3Aa and Figure Ba to exclude debris and dead cells. Group the cells within the gated region as population 1 (P1).
  3. To exclude cell clumps and doublets, plot P1 cells next based on the Side Scatter Width (SSC-W) versus the SSC-A. Gate the population of single cells as population 2 (P2) (Figure 3Ab and Figure Bb).
  4. Plot P2 cells based on the Forward Scatter Width (FSC-W) versus the FSC-A and gate the population of single cells as population 3 (P3) (Figure 3Ac and Figure Bc).
    NOTE: Use of two consecutive gates in 7.3 and 7.4 excludes cell clumps and doublets (high FSC-W and high FSC-A).
  5. Gate P3 cells based on GFP versus allophycocyanin (APC). The APC channel detects autofluorescence. Gating on this channel avoids capturing autofluorescent cells. Use GFP-negative cells to adjust the voltage for FITC/GFP fluorescent channels. Ideally, position gates for these cell populations around 102. Select gate thresholds for GFP-positive population 4 (P4) individually for each type of motor neuron (Figure 3Ad and Figure Bd).
    NOTE: Set the GFP gate much higher for SMNs than for CN3s/CN4s in order to obtain a pure culture (Figure 3Ad and Figure Bd). A lower GFP gate for SMN cultures leads to contamination of the cultures by glia and non-motor neurons. This is likely because there is low-level GFP expression in some glia and non-motor neurons due to a leaky promoter. The percentage of GFP-positive cells as compared to total cells is typically 0.5−1.5% for CN3s/CN4s and 1.5−2.5% for SMNs. If the dissection was successful, these numbers can be used as a benchmark to determine the appropriate position for the GFP-positive gate (Figure 3Ae and Figure Be).
  6. Perform FACS according to the manufacturer's protocol. Collect P4 cells into fresh 1.7 mL microcentrifuge tubes filled with 500 µL of motor neuron culture medium. Store on ice until plating.
    NOTE: Although the cells can be sorted directly into the wells, this results in an uneven number of cells per well. Sort the cells into 1.75 mL microcentrifuge tubes and then plate manually in order to achieve a more even plating distribution.

8. Culture of Purified Primary Motor Neurons

  1. Dilute FACS-isolated CN3/CN4 and SMN suspensions with motor neuron culture medium prewarmed to 37 °C to densities of 5 x 103 and 1 x 104 cells/mL, respectively.
    NOTE: One E11.5 embryo yields approximately 1 x 103 CN3/CN4 and 7 x 103 SMN. However, these yields rely heavily on the purity of dissected tissues, the thoroughness of cell dissociation, and the appropriate thresholding of GFP gates during FACS.
  2. Transfer 96 well plates precoated with PDL and laminin from the 37 °C tissue culture incubator to the laminar flow hood and aspirate laminin from each well. Use plates and coverslips immediately without washing.
  3. Add 200 µL of diluted CN3/CN4 and SMN suspensions into the each well of PDL/laminin-coated 96 well plates. Final cell densities should be 1 x 103 and 2 x 103 cells/well for CN3/CN4 and SMN, respectively.
    NOTE: Initial plating density of SMNs in 96 well plates (2 x 103 cells/well) is double that of CN3s/CN4s (1 x 103 cells/well) in order to obtain similar final motor neuron numbers and densities at 2 and 9 days in vitro (DIV) (4−6 x 102 and 2−4 x 102 cells per well, respectively).
  4. Culture neurons in a 37 °C, 5% CO2 incubator.
  5. Feed neurons every 5 days by removing half of the old media (100 µL) and replacing with the same volume of fresh motor neuron culture medium. Ensure that neuronal processes become visible on 1 DIV and become thicker and longer by 14 DIV (Figure 4). Neuronal cell bodies become enlarged and tend to aggregate in long-term cultures, particularly for SMNs (Figure 4).
    NOTE: Perform all medium and solution changes by leaving half of the original medium volume in order to avoid detaching cultured cells. This includes fixation and immunocytochemistry (ICC) steps. If all media is removed, regardless of how gently, most of the cells will detach and be washed away.

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Results

The aim of this protocol was to highly purify and culture both primary CN3s/CN4s and SMNs long-term to enable comparative analyses of the mechanisms underlying motor neuron disorders (see Figure 1 and Figure 2 for overview).

Once neurons were successfully isolated and grown in culture, nearly pure primary CN3/CN4 and SMN cultures were obtained (Figu...

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Discussion

Historically, in vitro studies of CN3 and/or CN4 motor neurons have relied on heterogeneous cultures such as dissociated17,18,19,20,21, explant17,22,23,24,25,26, a...

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Disclosures

The authors declare no conflict of interest.

Acknowledgements

We thank Brigitte Pettmann (Biogen, Cambridge, MA, USA) for instruction in SMN dissection techniques; the Dana Farber Cancer Institute Flow Cytometry Facility, the Immunology Division Flow Cytometry Facility of Harvard Medical School, The Joslin Diabetes Center Flow Cytometry Core, Brigham and Women's Hospital Flow Cytometry Core, and Boston Children's Hospital Flow Cytometry Research Facility for FACS isolation of primary motor neurons; A.A. Nugent, A.P. Tenney, A.S. Lee, E.H. Nguyen, M.F. Rose, additional Engle laboratory members, and Project ALS consortium members for technical assistance and thoughtful discussion. This study was supported by Project ALS. In addition, R.F. was funded by the Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad and NIH Training grant in Genetics T32 GM007748; J.J. was supported by the NIH/NEI training program in the Molecular Bases of Eye Diseases (5T32EY007145-16) through Schepens Eye Research Institute and by the Developmental Neurology Training Program Postdoctoral Fellowship (5T32NS007473-19) through Boston Children's Hospital; M.C.W was supported by NEI (5K08EY027850) and Children's Hospital Ophthalmology Foundation (Faculty Discovery Award); and E.C.E. is a Howard Hughes Medical Institute Investigator.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L)Thermo Fisher ScientificA-110011:400
Alexa Fluor 594-conjugated F(ab')2 goat anti-rabbit IgG (H+L)Thermo Fisher ScientificA-110721:400
B27 Supplement (50x), serum freeThermo Fisher Scientific17504-044
BD FACSAria llu SORP Flow CytometerBD Bioscience-This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers.
BD Falcon 70μm Nylon Cell StrainersCORNING352350For filtering the dissociating cells before FACS.
BD Falcon Round Bottom Test Tubes With Snap CapCORNING352054
BDNF HumanProSpec-Tany TechnoGene, Ltd.CYT-207
Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well)Greiner Bio-One International655090We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC
Circular Cover Glasses for microscopyKarl Hecht & Assistent1001/14We used this coverslip since the area was large (diamater: 14 mm).
CNTF HumanProSpec-Tany TechnoGene, Ltd.CYT-272
Cyclopiazonic acid from Penicillium cyclopiumSigma-AldrichC1530CPA. One of ER stressors.
4′,6-diamidino-2-phenylinodole (DAPI)Thermo Fisher ScientificD1306
Dimethyl sulfoxideSigma-AldrichD2650DMSO
Dumont #5 Forceps Inox Tip Size 0.05 mm x 0.01 mm Biologie TipsRoboz Surgical InstrumentRS-5015
ForskolinThermo Fisher ScientificBP25205
GDNF HumanProSpec-Tany TechnoGene, Ltd.CYT-305
GlutaMAX supplementThermo Fisher Scientific35050-061
Hanks’ Balanced Salt Solution (HBSS)Thermo Fisher Scientific14175-095
Hibernate EBrainBitsHE
Hibernate E low fluorescenceBrainBitsHELFFluorescence which hinders observation of embryo's GFP expressions should be low.
Horse serum, heat inactivated, New Zealand originThermo Fisher Scientific26050-070
IBMXTocris Cookson2845Isobutylmethylxanthine
LamininThermo Fisher Scientific23017-015
Leibovitz’s L15 mediumThermo Fisher Scientific11415064
2-MercaptoethanolSigma-AldrichM6250
Micro Dissecting ScissorsRoboz Surgical InstrumentRS-5913
Micro Knife 4.75" 1.7 mm x 27 mm bladeRoboz Surgical InstrumentRS-6272
Moria Mini Perforated SpoonFine Science Tools10370-19
Mouse monoclonal antibody to neuronal class III β-tubulin (TUBB3)BioLegend8012021:500, TUJ1
Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements softwareNikon-Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments
Nitric Acid 90%, Fuming (Certified ACS)Fisher ScientificA202-212For rinsing coverslips
Olympus 1.7 mL Microtubes, ClearGenesee Scientific22-281These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context.
Papain Dissociation SystemWorthington Biochemical CorpLK003150Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit.
Penicillin-streptomycin (10,000 U/mL)Thermo Fisher Scientific15140-122
Phosphate buffered saline (PBS)Thermo Fisher Scientific10010-023
Poly D-lysin (PDL)MilliporeSigmaA-003-E
Rabbit monoclonal antibody to Islet1Abcamab1095171:200
SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 cameraNikon-Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes.
Sylgard 170 Black Silicone Encapsulant - A+B 0.9 kg kitDow Corning1696157We make dissection dishes using this kit.
TC treated Dishes, 100 mm x 20 mmGenesee Scientific25-202We make dissection dishes using this dish.
Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip WidthRoboz Surgical InstrumentRS-8100
Transducer for LOGOQ e VETGE HealthcareL8-18i-RSFor ultrasound on female mice
Veterinary ultrasound machineGE HealthcareLOGOQ e VETFor ultrasound on female mice
Zeiss LSM 700 series laser scanning confocal microscope and Zen SoftwareCarl Zeiss-Confocal image of the embryo was captured with these equipments

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Oculomotor NeuronsTrochlear NeuronsIsl1 GFP MiceFACS Cell IsolationTissue DissectionPrimary Neuron CultureMotor Neuron PurificationEmbryonic Day 11 5GFP Positive Cells
Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice
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