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Aug 2018

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Primary Embryonic Rat Cortical Neuronal Culture and Chronic Rotenone Treatment in Microfluidic Culture Devices
原代胚胎大鼠皮质神经元培养并长期在微流控培养装置中鱼藤酮处理    

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Abstract

In the study of neurodegenerative diseases, it is imperative to study the cellular and molecular changes associated with pathogenesis in the relevant cell type, central nervous system neurons. The unique compartmentalized morphology and bioenergetic needs of primary neurons present complications for their study in culture. Recent microculture techniques utilizing microfluidic culture devices allows for environmental separation and analysis of neuronal cell bodies and neurites in culture. Here, we present our protocol for culture of primary neurons in microfluidic devices and their chronic treatment with the Parkinson’s disease (PD) relevant toxicant rotenone. In addition, we present a method for reuse of devices for culture. This culture methodology presents advantages for evaluating early pathogenic cellular and molecular changes in neurons in a compartment-specific manner.

Keywords: Primary neuron culture, Microfluidic culture, Microfluidic device, Microfluidic chamber, Rotenone, Parkinson’s disease, Microfluidic device reuse

Background

Studying primary neurons in vitro has long presented challenges due to the unique, diverse morphologies of neurons, and due to neuronal culturing requirements (Millet and Gillette, 2012). Neurons exhibit a unique compartmentalized morphology, with the soma, dendrites, and axons all exhibiting compartment-specific biochemical needs (Van Laar and Berman, 2013). Studying these different microenvironments objectively via large-scale approaches or high-throughput methods can be difficult under traditional plate culture, as cell bodies and neurites growing in proximity often cross, overlap, and functionally connect with one another. The development of microculturing methods incorporating microchannels to restrict cell movement and permit neurite outgrowth has allowed for the culturing of neurons in a manner that environmentally separates axonal and dendritic neurite projections from their cell body, or soma (Millet and Gillette, 2012; Taylor et al., 2003). This innovation permits cellular compartment-specific analyses of neuronal development, biochemistry, and effects of treatments (e.g., treating the axons with a drug or toxicant, but not the soma) (Taylor et al., 2005 and 2015; Park et al., 2006). The commercial availability of such devices now allows for uniform, large-scale studies into comparisons of the axonal environment and the somal environment.

By allowing for prolonged, healthy culture of primary neurons while isolating the microenvironments of the differing cellular compartments, these devices have become essential tools for studies that examine morphological-compartment specific development, senescence, or, in the case of our previous study, chronic drug exposure (Van Laar et al., 2018).

Here, we present our detailed protocol for preparation of embryonic rat primary cortical neurons (Arnold et al., 2011; adapted from Ghosh and Greenberg, 1995), culturing the neurons in Xona Microfluidics® brand microfluidic culture devices, and chronically treating the neurons with sublethal levels of the PD-relevant toxicant rotenone, as described in our recent publication in Van Laar et al. (2018). Our microfluidic device plating protocol, adapted from work originally presented by Park et al. (2006), provides details to (1) better ensure isolation of the soma from the axonal compartment side during the initial plating and (2) to promote neuronal health during prolonged culturing and treatment requiring media changes. We also present our protocol for the cleaning and reuse of microfluidic culture devices. This helps prevent the waste of devices in the event that initial platings of neuronal cultures fail to thrive or are otherwise unusable for experimental analyses.

Materials and Reagents

  1. Materials
    1. 6 cm sterile culture Petri dishes (Fisher, catalog number: AS-4052)
    2. 10 cm sterile culture Petri dishes (Corning, catalog number: 430167)
    3. 15 cm sterile culture Petri dishes (Corning, catalog number: 430599)
    4. 10 ml sterile syringes (BD, catalog number: 309604)
    5. PES 0.22 µm-pore sterile syringe filters (Millipore, catalog number: SLGP033RS)
    6. Sterile 0.22 µm Bottle-Top Filters (Fisher, catalog number: 09-761-112)
    7. Media Storage Bottles, 500 ml, 1,000 ml (Fisher, catalog numbers: 06-414-1C, 06-414-1D)
    8. Hydrion pH paper (Fisher, catalog number: 14-853-150N)
    9. 15 ml Falcon Conical Tubes (Fisher, catalog number: 14-959-70C)
    10. 50 ml Falcon Conical Tubes (Fisher, catalog number: 14-959-49A)
    11. 40 µm Sterile Cell Strainer (Fisher, catalog number: 22363547)

  2. Animal
    E18 Pregnant female Sprague-Dawley rat

  3. Primary neuron dissection
    1. Sodium sulfate (Na2SO4) (Sigma, catalog number: S5640-500G)
    2. Potassium sulfate (K2SO4) (Sigma, catalog number: P8541-1KG)
    3. Magnesium chloride (MgCl2), 1 M solution (Sigma, catalog number: M1028-100ML)
    4. Calcium chloride, anhydrous (CaCl2) (Sigma, catalog number: C5670-500G)
    5. HEPES, 1 M solution (Gibco, catalog number: 15630-080)
    6. Glucose (Gibco, catalog number: 15023-021)
    7. Phenol Red (Sigma, catalog number: P0290-100ML)
    8. Sodium Hydroxide (NaOH), 10 N (Ricca Chemical, catalog number: 7470-32)
    9. Cell-culture grade sterile water (Hyclone, catalog number: SH30529.03)
    10. Hanks’ Balanced Salt Solution (HBSS), 10x (Sigma, catalog number: H1641-500ML)
    11. Sodium bicarbonate (NaHCO3) (Sigma, catalog number: S5761-500G)
    12. L-Cysteine hydrochloride monohydrate (Cysteine-HCl) (Sigma, catalog number: C7880-100G)
    13. Papain (Worthington Biochemical Corp, catalog number: LS003126)
    14. Cell-culture grade sterile water (Hyclone, catalog number: SH30529.03)
    15. Bovine Serum Albumin (BSA), Fraction V (Fisher, catalog number: BP1600-100)
    16. Soybean Trypsin Inhibitor (Sigma, catalog number: T6522-1G)
    17. Phosphate Buffered Saline (PBS), 10x Solution (Fisher, catalog number: SH3037803)
    18. 1x PBS Solution (diluted from 10x PBS using cell-culture grade sterile water)
    19. Paraformaldehyde (PFA) (Fisher, catalog number: AC416780030)
    20. HBSS Dissection Media (store at 4 °C for up to 3 months) (see Recipes)
    21. Dissociation Media (store at 4 °C for up to 3 months) (see Recipes)
    22. L-Cysteine 50x Stock (see Recipes)
    23. Papain Dissociation Solution and Inhibitor Solutions for Isolation of Primary Rat Cortical Neurons (make fresh on the day of use) (see Recipes)
      1. Enzyme Solution
      2. Heavy Inhibitor Solution
      3. Light Inhibitor Solution
    24. 4% PFA Solution (see Recipes)

  4. Plating and culture media
    1. Neurobasal Media (Gibco, catalog number: 21103-049)
    2. Fetal Bovine Serum, Heat-Inactivated (Hyclone, catalog number: SH30070.03HI)
    3. B27 Media Supplement, 50x (Gibco, catalog number: 17504-044)
    4. Glutamax (Gibco, catalog number: 35050-061)
    5. Penicillin/Streptomycin Antibiotic Solution (Gibco, catalog number: 15140-122)
    6. Cortical Neuron Media for Seeding (store each at 4 °C for up to 10 days) (see Recipes)
    7. Cortical Neuron Media for Feeding (store each at 4 °C for up to 10 days) (see Recipes)

  5. Microfluidic device preparation, culture, and reuse
    1. Xona Microfluidics® microchannel culturing microfluidic devices
      1. 450 μm microgroove barrier Standard Neuron Device (Xona Microfluidics, catalog number: SND450)
      2. 900 μm microgroove barrier Standard Neuron Device (Xona Microfluidics, catalog number: SND900)
    2. Corning cover glass coverslip (Corning, catalog number: 2975-244)
    3. Poly-D-Lysine hydrobromide, MW 70,000-150,000 (Sigma, catalog number: P0899-50MG)
    4. Boric Acid (Fisher, catalog number: S79802)
    5. Borax, Anhydrous (MP Biomedicals, catalog number: 190309)
    6. HCl, 12 N (Fisher, catalog number: A144-500)
    7. Cell-culture grade sterile water (Hyclone, catalog number: SH30529.03)
    8. RBS detergent solution (Thermo, catalog number: 27950)
    9. 100% Ethanol (Decon Labs, catalog number: 2705HC)
    10. 0.1 M Borate Buffer Solution (see Recipes)
    11. Poly-D-Lysine 2.5 mg/ml Stock Solution (see Recipes)
    12. Poly-D-Lysine 100 µg/ml Coating Solution (see Recipes)

  6. Rotenone treatment
    1. DMSO (Sigma, catalog number: D2438)
    2. Rotenone (Sigma, catalog number: R8875-1G)
    3. Rotenone Stock Solution (see Recipes)
    4. Rotenone 2x and 1x solutions (see Recipes)
    5. DMSO Vehicle Control 2x and 1x solutions (see Recipes)

Equipment

  1. CO2 tank 
  2. CO2 single-stage flowmeter regulator (Western Medica, model: M1-320-12FM)
  3. CO2 gas anesthetizing Box (Braintree Scientific, model: AB-2)
  4. Motic Dissection microscope (Motic, model: SMZ-168)
  5. Roboz surgical instrument dissection tools 
    1. Dumont #5 Forceps, Straight tip (Roboz, catalog number: RS-5060)
    2. Dumont #5 Forceps, 45 Degree tip (Roboz, catalog number: RS-5005)
    3. Straight, Sharp-Blunt Operating Scissors (Roboz, catalog number: RS-6812)
    4. Extra Fine Micro Dissecting Scissors (Roboz, catalog number: RS-5882)
    5. Micro Dissecting Spring Scissors (Roboz, catalog number: RS-5603)
  6. Sorvall Legend T table-top centrifuge (Thermo, model: 75004367)
  7. Nuaire Class II Type A2 Laminar-flow sterile culture hood with vacuum waste system (Nuaire, model: NU-425-400)
  8. Leica Fiberlight light source (Leica, model: EEG2823)
  9. Olympus light microscope (Olympus, model: CKX31)
  10. HeraCell CO2- and Temperature-Controlled culture incubator (Thermo, model: 51022394)
  11. Isotemp 110 Heated Water Bath (Fisher Scientific, model: 15-460-10)
  12. Wheaton Glass Slide Staining Jar (Fisher Scientific, model: 22-309-247)
  13. Disposable transfer pipettes (Fisher Scientific, catalog number: 13-711-20)
  14. Rocking Shaker Plate (Labnet, model: S-2035)
  15. Heated Stir Plate (Corning, model: PC-420)
  16. Accumet basic pH meter (Fisher Scientific)
  17. Vortex Mixer (Labnet, model: VX100)
  18. Scale (Denver Instruments, model: APX-100)
  19. Millipore Milli-Q Ultrapure Water System
  20. Assorted mechanical pipettes and corresponding sterile tips
  21. Hemocytometer and Cell Counter
  22. Thermometer
  23. Autoclave
  24. Chemical Fume Hood
  25. 4 °C refrigerator
  26. -20 °C freezer
  27. -80 °C freezer

Procedure

  1. Prepare in advance of dissection and plating
    1. Prepare Borate Buffer solution, the Dissociation Media and the HBSS Dissection Media according to the Recipes below.
    2. Prepare the necessary amounts of Seeding Media and Feeding Media according to the Recipes below. The amount prepared will depend on the size of the planned experiment. 
      1. Each full neuronal preparation needs approximately 25 ml of Seeding Media, and each device needs approximately 5 ml of Feeding Media for plating and maintenance.
      2. Prepared media can be kept for up to 10 days, and could be used for subsequent neuronal preparations. It is therefore reasonable to make enough media for subsequent uses. 

  2. Prepare 2-days before dissection and plating
    1. Thaw Poly-D-Lysine
      1. Pull the necessary number of aliquots of 2.5 mg/ml Poly-D-Lysine Stock Solution (400 µl) from -80 °C.
      2. Place on ice, and thaw overnight at 4 °C.
    2. Acid-wash and sterilize coverslips
      1. In a sterile culture hood, place the required number of coverslips (1 per device to be plated, plus 2 extras) into a glass slide staining jar.
      2. Fill with 50 ml of 1 N HCl (prepared in sterile cell culture water). 
      3. Place the cover on the staining jar, and place the jar on a shaker plate outside the hood. Agitate the coverslips gently on a shaker plate for 1 h.
      4. Return the jar to the sterile culture hood and remove the HCl from the jar via pipette or aspiration.
      5. Add sterile cell culture water to the jar, cover, and place the jar on a shaker plate outside the hood. Repeat this water rinse 3 x 10 min.
      6. Remove final water wash via pipette or aspiration, and add 50 ml of 70% ethanol solution to sterilize coverslips. Cover the jar and agitate gently on a shaker plate for 30 min.
      7. Remove ethanol wash via pipette or aspiration, and add 50 ml of sterile culture water to the coverslips. Cover the jar and agitate gently on a shaker plate for 10 min.
      8. In the sterile culture hood, remove coverslips from the staining jar, and rest in an open 10 cm Petri dish to air dry. Prop the edges of the coverslips up on the sides of the petri dish–do not let the coverslips lay flat.
      9. Once dry, sterilize the coverslips by exposing them to the ultraviolet (UV) light of the sterile culture hood for 20 min.
      10. After UV exposure, place the coverslips inside the Petri dish and cover with the lid. Store overnight in a closed container or in the sterile culture hood without the UV light on.

  3. Prepare 1-day before dissection and plating
    1. Coat coverslips with Poly-D-Lysine
      1. Carry this procedure out in a sterile culture hood.
      2. Arrange coverslips, so they are lying flat, not overlapping, on the bottom of a 10 cm Petri dish.
      3. Prepare 6 ml of 100 µg/ml Poly-D-Lysine Coating Solution per 10 cm dish by adding the 2.5 mg/ml Poly-D-Lysine Stock Solution to sterile 0.1 M Borate Buffer in the ratio of 400 µl, 2.5 mg/ml Poly-D-Lysine stock to 9.6 ml, 0.1 M Borate Buffer.
      4. Mix and sterile filter the 100 µg/ml Poly-D-Lysine Coating Solution.
      5. Cover the coverslips with the 100 µg/ml Poly-D-Lysine Coating Solution, place the lid on the Petri dish, and allow the solution to coat coverslips overnight at 4 °C.
    2. Sterilize microfluidic devices
      1. Working in a sterile culture hood, remove devices from packaging.
      2. Place devices groove-side up (i.e., upside-down) in a sterile 10 cm Petri dish.
      3. Submerse the devices in 100% ethanol for 30 min.
      4. Remove ethanol, and submerse devices in 70% ethanol for 30 min.
      5. Remove devices from ethanol, and submerse in a dish of sterile cell culture water.
      6. Set devices at an angle on the edge of a sterile Petri dish and allow to fully dry in the sterile culture hood.
      7. Place in the Petri dish and cover. Store overnight in a closed container or in the sterile culture hood in the dark, without either UV light or the fluorescent light on.
      8. Do not expose the devices to UV light at any time.

  4. Day of dissection
    1. Prepare papain solution and inhibitor solutions according to the Recipes below.
    2. Place Feeding Media, Seeding Media, the Papain Digestion Solution, and the papain inhibitor solutions in a 37 °C water bath.
    3. Place HBSS Dissection Solution on ice. Aliquot approximately 150 ml HBSS to be used at the bench, and 25 ml HBSS to remain sterile for use in the hood.
    4. Rinse and dry coverslips
      1. Working in a sterile culture hood, use sterile forceps to remove coverslips from the Poly-D-Lysine solution and place angled on the edge of a sterile Petri dish to dry.
      2. Once dry, dip each coverslip 3 x in sterile culture water to rinse, allowing water to drip off between dips.
      3. Dip the coverslip in sterile 1x PBS, let solution drip off, and return to the Petri dish, propped up against the edge of the dish and allowed to dry fully. Do not expose to UV light.
    5. Attach microfluidic devices to coverslips and prime with Feeding Media–see Figure 1 for device placement and orientation
      1. Carry this procedure out in a sterile culture hood.
      2. Using sterile forceps, place a single Poly-D-Lysine-coated coverslip onto the bottom of a sterile 6 cm Petri dish, making sure to have the Poly-D-Lysine-coated side up.
      3. Using a second set of sterile forceps, lift a microfluidic device and place it, microgroove-side-down, onto the center of the coverslip. (Figure 1A)
      4. Use the blunt end of the forceps to gently press the device onto the coverslip, making sure to gently press out any air bubbles to ensure a tight seal.
      5. Place the lid on the Petri dish to maintain sterility, and examine the device assembly under a light microscope to ensure a tight seal around the microgrooves and feeding reservoirs. 
      6. Return to the sterile culture hood and prime the device with warmed Feeding Media as follows (Figures 1B and 1C):
        1. Add 100 µl of warm Feeding Media to the top-left reservoir. Watch to make sure the media enters and fills the channel connecting the two left reservoirs.
        2. Wait 2 min to ensure the media has filled the axonal channel and the microgrooves.
        3. Next, add 100 µl of warm media to the top-right reservoir.
        4. Wait 2 min to ensure the media has filled the somal channel.
        5. Add 100 µl of warm media each to the bottom-left and bottom-right reservoirs.
        6. Place the lid on the Petri dish holding the device, and place in a cell culture incubator (37 °C, 5% CO2) for at least 1 h before plating neurons.


      Figure 1. Schematic of microfluidic device mounting, orientation, and priming with culture media. A. Placement of microfluidic device on coverslip. B. Device features and orientation. C. Order of media addition to prime device.

    6. Primary cortical neuron dissection and cell preparation
      At the bench
      1. An E18 pregnant female rat is humanely sacrificed and embryonic rat primary cortices dissected (meninges removed) per established lab methods and according to an approved animal protocol adhering to the requirements of the University of Pittsburgh Department of Laboratory Animal Research (DLAR) and Institutional Animal Care and Use Committee (IACUC). These requirements will vary between institutions. Our methods were adapted from previous work as described by Ghosh and Greenberg (1995), and as previously described by our lab (Arnold et al., 2011). Cell yield varies and is approximately 0.5-1.0 x 106 cells per embryo.
      2. Gently transfer all cortices to a 15 ml conical tube on ice containing 9 ml HBSS dissection media.
      3. After the dissections are complete, move to a sterile culture hood to continue the procedure.

      In sterile culture hood
      1. Gently remove HBSS from cortices using a transfer pipette and discard HBSS.
      2. Gently wash cortices 3 x using 5 ml cold HBSS for each wash. After each wash, gently remove HBSS from cortices using a transfer pipette and discard.
      3. After the last HBSS wash, promptly add 5 ml of pre-warmed Enzyme Solution to the cortices and incubate at 37 °C for 20 min.
      4. Remove Enzyme Solution using a transfer pipette and immediately add the pre-warmed Light Inhibitor Solution (10 ml) to cortices for 1 min.
      5. Remove the Light Inhibitor Solution using a transfer pipette and immediately add the Heavy Inhibitor Solution (5 ml) to cortices for 2 min at 37 °C.
      6. Remove the Heavy Inhibitor Solution using a transfer pipette and gently wash the cortices 3 x using 5 ml of 37 °C Seeding Media for each wash. After each wash, gently remove media from cortices using a transfer pipette and discard.
      7. Add fresh 5 ml Seeding Media to the cortices and use a P1000 (1 ml) mechanical pipette and triturate by pipetting up-and-down approximately 20 times to break up the tissue and cell clumps into a homogeneous cell suspension. You may need to triturate for longer (5-10 more times) if the cells are still in clumps, but do not triturate excessively. The cell suspension should look cloudy and there should be no obvious large particles or cell clumps remaining. Alternatively, the cells can be run through a 40 µm sterile cell strainer sieve after the 20-times trituration.
      8. Count cells
        1. Combine a 10 µl aliquot of suspended cells with 90 µl of Seeding Media.
        2. Use hemocytometer to count cells and calculate cells/ml in suspension.

  5. Plating microfluidic devices
    Primary cortical neurons are plated at 50,000 cells per device for standard 2-compartment microfluidic neuron devices with 450 µm- or 900 µm-width microgroove barriers (Xona microfluidics). To help ensure that the plating doesn’t result in cells being forced through the microgrooves to the axonal, non-cell side of the device, proceed as follows (Figure 2):
    1. Adjust cell suspension to 2.5 x 106 cells/ml in Seeding Media. Prepare enough for 20 µl of cell solution per device to be plated. Set aside.
    2. Remove the Petri dishes holding mounted microfluidic devices from the incubator, and place them in the sterile culture hood.
    3. Remove all of the media used to prime the device from all reservoirs.
    4. Add 100 µl of Feeding Media to the top-left reservoir (axonal side) of each device.
    5. Add 100 µl of Feeding Media to the connected bottom-left reservoir (axonal side). Wait 1 min for media to equilibrate on the axonal side of the device. (see Notes)
    6. On the somal side of the device, add 10 µl of cell suspension to the top-right reservoir (somal side).
      Note: Place the 10 µl cell suspension at the junction of where the reservoir joins the channel (see Figure 2).
    7. Cover the Petri dish holding the device, and examine under a light microscope to ensure that cells are entering the channel between the top-right and bottom-right reservoirs. (see Notes)
    8. After 1 min, Add another 10 µl of cell suspension to the connected bottom-right reservoir (somal side).
    9. Place the lid on the Petri dish holding the device and return it to the incubator. Allow 10 min for the cells to attach.
    10. After 10 min, remove the device from the incubator and return to the sterile culture hood.
    11. Gently add an extra 100 µl of Feeding Media to the top-left reservoir (axonal side).
    12. Gently add an extra 100 µl of Feeding Media to the connected bottom-left reservoir (axonal side).
    13. Immediately after Step E12, gently add 200 µl of Feeding Media to the cell-side top-right reservoir (somal side). 
    14. Immediately following, gently add 200 µl of Feeding Media to the connected bottom-right reservoir (somal side).
      Notes:
      1. Be slow and gentle when adding media; adding media forcefully or too fast will wash the cells out of the channel.
      2. Now each reservoir on the left has 200 µl of Feeding Media, and each reservoir on the right has 210 µl of Feeding Media.
    15. Place the lid on the 6 cm Petri dish holding the device. 
    16. Place the covered 6 cm Petri dishes holding devices into a 10 cm Petri dish. Up to three 6 cm dishes will fit into one 10 cm dish. Place the lid on the 10 cm Petri dish and return devices to the incubator. This dish-within-a-dish system is used to help reduce evaporation and maintain media levels in the devices during the culture period.


      Figure 2. Schematic of seeding neurons into the microfluidic device

  6. Feeding maintenance and rotenone treatment
    1. Maintain cells
      1. After plating, add 10 µl of Feeding Media to each of the four reservoirs on each device every 3-4 days to maintain media levels. Add warmed media to devices in the sterile culture hood.
      2. No media changes occur until the initial treatment.
    2. Chronic rotenone and DMSO vehicle treatment
      On day in vitro 7 (DIV7), add the initial treatment with rotenone or DMSO vehicle control, via a ½ media change in both the axonal side and somal side reservoirs as follows: 
      1. Prepare a 2x concentration of the desired final rotenone concentration in warm Feeding Media (see Recipes for solution preparation).
      2. In parallel, prepare warm Feeding Media with a corresponding 2x concentration of DMSO as a vehicle control (see Recipes for solution preparation).
      3. Remove the Petri dish holding microfluidic devices from the incubator.
      4. In the sterile culture hood, remove 100 µl of media from each of the four reservoirs on each device.
      5. For each device, add 100 µl of the 2x rotenone or 2x DMSO solution to each of the four reservoirs.
      6. The final concentration will be 1x rotenone or 1x DMSO throughout the device.
    3. Treatments continue for 1-2 weeks with ½ media changes every 3-4 d using 1x concentration media.
      1. Prepare a 1x concentration of the desired final rotenone concentration and a 1x concentration of DMSO vehicle control each in warm Feeding Media (see Recipes for solution preparation).
      2. Repeat the ½ media change for each reservoir as described above.

  7. Fixing neurons (Figure 3)
    1. At the end of the experiment, remove media from all reservoirs.
    2. Immediately add 100 µl of warm 1x PBS to the top-left and top-right reservoirs.
    3. Allow PBS to flow to bottom reservoirs for 1 min.
    4. Immediately remove all PBS.
    5. Add 100 µl of warm 4% PFA into each top reservoir.
    6. Allow cells to fix for 20 min at room temperature.
    7. Remove all PFA, and dispose of properly. 
    8. Add 200 µl of PBS to each top reservoir.
    9. Rinse for 5 min.
    10. Remove PBS rinse.
    11. Repeat PBS rinse 2 x.
    12. Fill each reservoir with PBS.
    13. Cover device, and store at 4 °C until use for further analyses. 


    Figure 3. Schematic of fixing neurons in the microfluidic device

  8. Reusing microfluidic devices
    In the event that a culture dies before experimental treatments begin, the device was not appropriately mounted and cells entered the non-cell-body side of the device, or the culture is otherwise unusable for experiments, we have found that the device can be reused successfully. Do not reuse devices that have been exposed to experimental treatments, such as drugs or toxicants (see Notes). Starting from a mounted microfluidic device with an active culture, follow the following steps for device reuse:
    1. Remove the media from the device reservoirs using a pipette. 
    2. Place a finger on the corner of the coverslip, then use forceps to gently grip the edge of the device at a reservoir and gently pull up at an angle until the device is free from the coverslip.
    3. Rinse the device gently with distilled water, then place groove-side-up in a Petri dish.
    4. Submerge the device in 1x RBS detergent solution. Cover the dish, and gently agitate on a shaker overnight at room temperature.
    5. Remove the RBS detergent and wash the device using culture grade water 3 x for 1 h each, with gently agitation on a shaker at room temperature.
    6. After the final wash, with the device still submerged in water, examine the device under a light microscope to verify that all cellular debris has been removed from the reservoirs and grooves.
    7. Remove the final wash, and carry out the remaining procedure in a sterile culture hood.
    8. Transfer the device, groove side up, to a sterile Petri dish, then submerge the device in sterile culture grade water. Cover the dish, and gently agitate on a shaker overnight at room temperature.
    9. In the sterile culture hood, remove the device from the sterile wash and either air dry in a sterile culture dish to store for future use, or carry out the sterilization procedure as listed above under Step C2 for immediate use.

Data analysis

Prior to use for experiments, data acquisition, and data analyses, carefully examine the devices and culture quality. Device cultures in which the cells appear in obvious distress prior to treatments should not be used for experiments or further analyses. Despite the design of the devices being such that cells should not be able to cross the microchannels, some cells occasionally do cross the microchannels. Glia in particular are able to readily cross. An occasional neuronal soma is not a concern. If after a week of culture you notice there are large populations of neuron somata in the axonal side, discontinue use of this device culture. If after your experiment you notice many somata on the axonal side, do not use this device culture for data analyses. Large numbers of somata on the axonal side may indicate there were issues with plating (see Notes) or that the device was not properly attached to the coverslip.

Notes

Plating neurons in microfluidic devices (Procedure E)

  1. The plating of the cells is a crucial step to maintaining the isolation between soma and axons. Be sure that the reservoirs and channel on the axonal compartment side of the device are filled with Feeding Media 1-2 min before adding cells to the reservoirs on the somal compartment side of the device. If the axonal compartment is empty when cells are added, the fluid dynamics will force the detached cells through the microchannels and into the axonal channel space. If this occurs, the device and its neurons cannot be used for further experimentation as the axons will no longer be an isolated population. (Steps E2-E3)
  2. In Steps E4-E6, when adding the cells to the somal side of the device, it is advised to always check that cells are entering the somal compartment side channel via microscope. Fluid dynamics should pull the cells into the channel. If you do not observe this, return to the sterile culture hood and try gently removing the cells with a pipette. Make sure all media is removed from both top-right and bottom-right reservoirs, and again try placing the cells in the top-right reservoir where the reservoir joins the channel.

Reuse of microfluidic devices (Procedure H)
  1. Only reuse devices that have not been exposed to any exogenous treatment not typically found in culture media, such as drugs, toxicants (e.g., rotenone), or tissue fixative such as PFA. If the device has already been used during a treatment experiment or if the cells and device were already exposed to fixative, the device cannot be reused. Thus, any device in which an experiment was completed cannot be reused for another experiment.
  2. This reuse method only applies to microfluidic devices that were pressed onto an extracellular-matrix coated coverslip. If the devices were bound to the coverslip using plasma bonding techniques (see the manufacturer’s instructions), they cannot be reused.
  3. In our studies, we found that cultures from new devices and reused devices did not exhibit any discernable differences. Further, we observed no differences in the results obtained from experiments completed in new devices and experiments completed in reused devices.

Recipes

  1. 0.1 M Borate Buffer Solution
    1. 1.24 g boric acid and 1.002 g anhydrous borax in 400 ml cell culture grade sterile H2O
    2. Cover and stir on a stir plate overnight at RT
    3. Adjust pH to 8.4
    4. In sterile culture hood, filter sterilize into autoclaved media bottle
    5. Store at 4 °C for up to 3 months
  2. Poly-D-Lysine 2.5 mg/ml Stock Solution
    1. Thaw frozen lyophilized powder overnight at 4 °C on ice
    2. Prepare the stock in a sterile culture hood
    3. Add 20 ml of 0.1 M sterile Borate Buffer solution to 50 mg Poly-D-Lysine
    4. In sterile culture hood, filter sterilize the stock solution using a 10 ml syringes and 0.22 µm PES syringe filter
    5. Aliquot 400 µl stocks into sterile microfuge tubes
    6. Store at -80 °C
  3. Poly-D-Lysine 100 µg/ml Coating Solution
    1. Thaw an aliquot of Poly-D-Lysine Stock Solution (400 µl, 2.5 mg/ml) overnight at 4 °C on ice
    2. In sterile culture hood, add to 9.5 ml sterile 0.1 M Borate Buffer
    3. Final concentration: 100 µg/ml Poly-D-Lysine
  4. Cortical Neuron Media for Seeding
    Reagent
    % vol/vol
    For 500 ml
    Neurobasal Media
    91.5%
    457.5 ml
    B27 supplement
    2%
    10 ml
    Glutamax 100x
    1%
    5 ml
    Pen/Strep
    0.5%
    2.5 ml
    FBS
    5%
    25 ml
    Note: Prepare all the solutions in a sterile culture hood. 
    1. Combine all the reagents in a sterile culture hood, filter sterilize into an autoclaved media bottle
    2. Store at 4 °C for up to 10 days
  5. Cortical Neuron Media for Feeding
    Reagent
    % vol/vol
    For 500 ml
    Neurobasal Media
    96.5%
    457.5 ml
    B27 supplement
    2%
    10 ml
    Glutamax 100x
    1%
    5 ml
    Pen/Strep
    0.5%
    2.5 ml
    Note: Prepare all the solutions in a sterile culture hood.
    1. Combine reagents in a sterile culture hood, filter sterilize into an autoclaved media bottle
    2. Store at 4 °C for up to 10 days
  6. HBSS Dissection Media
    Reagent
    Amt.
    Final Conc.
    10x HBSS
    100 ml
    1x
    NaHCO3  350 mg
    4.2 mM
    HEPES, 1 M
    10 ml
    10 mM
    Glucose
    4.3 g
    35 mM
    Bring to 1 L with cell culture grade sterile H2O
    Notes:
    1. Do not adjust pH.
    2. Prepare all the solutions in a sterile culture hood.
    1. Combine reagents in a sterile culture hood, filter sterilize into an autoclaved media bottle.
    2. Store at 4 °C for up to 3 months
  7. Dissociation Media
    Reagent
    Amt.
    Na2SO4, 1 M
    20.44 ml
    K2SO4, 0.5 M
    15 ml
    MgCl2, 1 M 1.45 ml
    CaCl2, 100 mM
    0.625 ml
    HEPES, 1 M
    250 µl
    Glucose, 1 M
    5 ml
    Phenol Red, 0.5%
    0.5 ml
    NaOH, 0.1 N
    0.5 ml
    Bring to 250 ml with cell culture grade sterile H2O
    Notes:
    1. Do not adjust pH.
    2. Prepare all the solutions in a sterile culture hood.
    1. Combine reagents in a sterile culture hood, filter sterilize into an autoclaved media bottle
    2. Store at 4 °C for up to 3 months
  8. L-Cysteine 50x Stock
    1. Weigh out 160 mg, then prepare in sterile culture hood
    2. Dissolve in 10 ml of sterile Dissociation media
    3. Aliquot in sterile microcentrifuge tubes at 225 µl and store at -80 °C
  9. Papain Dissociation Solution and Inhibitor Solutions for Isolation of Primary Rat Cortical Neurons
    Notes:
    1. Make these solutions fresh on the day of use.
    2. Begin by aliquoting dissociation media into sterile 15 ml conical tubes in the sterile culture hood for each solution below.
    3. Prepare solutions at the bench with pH meter.
    4. After solutions are prepared, return to the sterile culture hood.
    5. For each solution, use a 10 ml sterile syringe and a syringe filter to sterile-filter the solution into a new, sterile 15 ml conical tube.
    6. Following preparation and final pH adjustment, filter-sterilize all solutions into new, sterile 15 ml conical vials under the sterile culture hood using 10 ml syringes and 0.22 µm PES syringe filters.
    1. Enzyme Solution
      10 ml Dissociation Media
      0.2 ml of 50x Cysteine-HCl
      200 U of Papain Solution
      Pulse on a vortex mixer
      Adjust pH to 7.4 with 0.1 N NaOH
    2. Heavy Inhibitor Solution
      6 ml Dissociation Media
      Add 60 mg BSA (4 °C) to 6 ml of DM
      60 mg of Soybean Trypsin Inhibitor (4 °C)
      Pulse on a vortex mixer, then place on a shaker to mix until fully in solution
      Adjust pH to 7.4 with 0.1 N NaOH
    3. Light Inhibitor Solution
      9 ml Dissociation Media
      Add 1 ml of Heavy Inhibitor Solution
      Pulse on a vortex mixer
      Adjust pH to 7.4 with 0.1 N NaOH
  10. Rotenone Stock Solution
    1. Use fresh rotenone and DMSO to prepare stocks
    2. Prepare 5 mM rotenone in DMSO 
    3. Aliquot into 100 µl aliquots and store at -20 °C
    4. Prepare enough stocks for all the experiments
  11. Rotenone 2x and 1x solutions
    1. Make fresh on the day of use, immediately before use
    2. Prepare in sterile culture hood
    3. Thaw an aliquot of 5 mM stock rotenone at RT protected from light
    4. Dilute directly into neuron Feeding Media to 2x of the final desired treatment concentration for the first treatment, and to 1x for subsequent treatments
  12. DMSO Vehicle Control 2x and 1x solutions
    1. Make fresh on the day of use, immediately before use
    2. Prepare in sterile culture hood
    3. Dilute DMSO directly into neuron Feeding Media to 2x of the final desired treatment concentration for the first treatment, and to 1x for subsequent treatments
    4. Vehicle control media should be prepared to have the same final concentration of DMSO as in rotenone-treatment media
  13. 4% PFA Solution
    To prepare 2 L of 4% PFA
    200 ml of 10x PBS
    80 g Paraformaldehyde
    Milli-Q Ultrapure Water to 2 L
    1. Prepare in a chemical fume hood 
    2. Add 1 L of ultrapure water to a 2 L flask with large stir bar
    3. Add 112.5 µl of 10 N NaOH
      Notes:
      i. Paraformaldehyde solubilizes in base better than in acid.
      ii. Leave out PBS at this stage to eliminate buffering.
    4. Suspend a thermometer in solution and place flask on a heated stir plate and stir
    5. Bring the solution to just below 60 °C and continue to stir until PFA is solubilized
      Note: Monitor closely; do not let the paraformaldehyde solution go above 60 °C .
    6. When PFA is solubilized solution pH to 7.4 with pH paper
      Note: Do not use pH meter . Adjust pH as necessary with HCl and NaOH.
    7. Add 200 ml of 10x PBS
    8. Bring the solution to 2 L with ultrapure water
    9. Recheck pH with pH paper to confirm that the final pH is 7.4
    10. Filter sterilize and prepare 40 ml aliquots in 50 ml conical tubes
    11. Store at -20 °C until use

Acknowledgments

This work was supported by a grant from the NIH (R01NS077954, S.B.B.) and a University of Pittsburgh Physicians Academic Foundation Research Grant (S.B.B).
The microfluidic device plating and culture methods were adapted from previous work as described by Park et al. (2006).
The chronic, low-dose rotenone exposure treatment methods were modified from our previous work as described in Arnold et al. (2011).

Competing interests

The authors have no conflicts of interest to disclose.

Ethics

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and are in accordance with guidelines put forth by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals. All euthanasia methods are consistent with the most updated American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.

References

  1. Arnold, B., Cassady, S. J., VanLaar, V. S. and Berman, S. B. (2011). Integrating multiple aspects of mitochondrial dynamics in neurons: age-related differences and dynamic changes in a chronic rotenone model. Neurobiol Dis 41(1): 189-200.
  2. Ghosh, A. and Greenberg, M. E. (1995). Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268(5208): 239-247.
  3. Millet, L. J. and Gillette, M. U. (2012). Over a century of neuron culture: from the hanging drop to microfluidic devices. Yale J Biol Med 85(4): 501-521.
  4. Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W. and Jeon, N. L. (2006). Microfluidic culture platform for neuroscience research. Nat Protoc 1(4): 2128-2136.
  5. Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W. and Jeon, N. L. (2005). A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2(8): 599-605.
  6. Taylor, A. M., Menon, S. and Gupton, S. L. (2015). Passive microfluidic chamber for long-term imaging of axon guidance in response to soluble gradients. Lab Chip 15(13): 2781-2789.
  7. Taylor, A. M., Rhee, S. W., Tu, C. H., Cribbs, D. H., Cotman, C. W. and Jeon, N. L. (2003). Microfluidic multicompartment device for neuroscience research. Langmuir 19(5): 1551-1556.
  8. Van Laar, V. S. and Berman, S. B. (2013). The interplay of neuronal mitochondrial dynamics and bioenergetics: implications for Parkinson's disease. Neurobiol Dis 51: 43-55.
  9. Van Laar, V. S., Arnold, B., Howlett, E. H., Calderon, M. J., St Croix, C. M., Greenamyre, J. T., Sanders, L. H. and Berman, S. B. (2018). Evidence for compartmentalized axonal mitochondrial biogenesis: mitochondrial DNA replication increases in distal axons as an early response to Parkinson's disease-relevant stress. J Neurosci 38(34): 7505-7515.

简介

在神经退行性疾病的研究中,必须研究与相关细胞类型,中枢神经系统神经元的发病相关的细胞和分子变化。 原代神经元的独特区室化形态和生物能量需求为其在培养中的研究提供了并发症。 最近利用微流体培养装置的微培养技术允许环境分离和分析培养物中的神经元细胞体和神经突。 在这里,我们提出我们的微流体装置中的原代神经元培养方案及其与帕金森病(PD)相关毒性鱼藤酮的长期治疗。 此外,我们提出了一种重用文化设备的方法。 该培养方法为以隔室特异性方式评估神经元中的早期致病细胞和分子变化提供了优势。
【背景】由于神经元的独特,多样形态以及神经元培养要求(Millet和Gillette,2012),研究体外原代神经元长期存在挑战。神经元表现出独特的区室化形态,体细胞,树突和轴突均表现出特定于细胞室的生化需求(Van Laar和Berman,2013)。在传统的平板培养下,通过大规模方法或高通量方法客观地研究这些不同的微环境可能是困难的,因为在附近生长的细胞体和神经突经常彼此交叉,重叠和功能性连接。包含微通道以限制细胞运动并允许神经突向外生长的微培养方法的发展允许以对环境上分离轴突和树突神经突突起与其细胞体或体细胞的方式培养神经元(Millet和Gillette,2012; Taylor 等人,2003)。这项创新允许细胞室特异性分析神经元发育,生物化学和治疗效果(例如,用药物或毒物治疗轴突,但不用躯体治疗)(Taylor et al。 ,2005和2015; Park et al。,2006)。现在,这种装置的商业可用性允许对轴突环境和正常环境进行比较的统一,大规模研究。

通过允许长时间,健康的原代神经元培养,同时隔离不同细胞区室的微环境,这些装置已经成为研究形态 - 隔室特异性发育,衰老的研究的必要工具,或者,在我们之前的研究中,慢性药物暴露(Van Laar et al。,2018)。

在这里,我们提出了制备胚胎大鼠原代皮层神经元的详细方案(Arnold et al。,2011;改编自Ghosh和Greenberg,1995),在Xona Microfluidics ®<中培养神经元/ sup>品牌微流体培养装置,并用亚致死水平的PD相关毒性鱼藤酮长期治疗神经元,如我们最近在Van Laar 等人(2018)中的出版物中所述。我们的微流体装置电镀方案改编自Park et al。(2006)最初提出的工作,提供了以下细节:(1)更好地确保在初始电镀期间从轴突室侧隔离躯体和( 2)在需要更换培养基的长期培养和治疗期间促进神经元健康。我们还提出了清洁和重复使用微流体培养设备的协议。这有助于防止在神经元培养物的初始平板未能茁壮成长或无法用于实验分析的情况下浪费设备。

关键字

材料和试剂

  1. 材料
    1. 6厘米无菌培养培养皿(Fisher,目录号:AS-4052)
    2. 10厘米无菌培养培养皿(Corning,目录号:430167)
    3. 15厘米无菌培养培养皿(Corning,目录号:430599)
    4. 10毫升无菌注射器(BD,目录号:309604)
    5. PES0.22μm孔无菌注射器过滤器(Millipore,目录号:SLGP033RS)
    6. 无菌0.22μm瓶顶过滤器(Fisher,目录号:09-761-112)
    7. 介质存储瓶,500毫升,1,000毫升(Fisher,目录号:06-414-1C,06-414-1D)
    8. Hydrion pH纸(Fisher,目录号:14-853-150N)
    9. 15毫升Falcon锥形管(Fisher,目录号:14-959-70C)
    10. 50毫升Falcon锥形管(Fisher,目录号:14-959-49A)
    11. 40μm无菌细胞过滤器(Fisher,目录号:22363547)

  2. 动物
    E18怀孕雌性Sprague-Dawley大鼠

  3. 原发性神经元解剖
    1. 硫酸钠(Na 2 SO 4 )(Sigma,目录号:S5640-500G)
    2. 硫酸钾(K 2 SO 4 )(Sigma,目录号:P8541-1KG)
    3. 氯化镁(MgCl 2 ),1M溶液(Sigma,目录号:M1028-100ML)
    4. 无水氯化钙(CaCl 2 )(Sigma,目录号:C5670-500G)
    5. HEPES,1 M解决方案(Gibco,目录号:15630-080)
    6. 葡萄糖(Gibco,目录号:15023-021)
    7. 酚红(Sigma,目录号:P0290-100ML)
    8. 氢氧化钠(NaOH),10 N(Ricca Chemical,目录号:7470-32)
    9. 细胞培养级无菌水(Hyclone,目录号:SH30529.03)
    10. 汉克斯平衡盐溶液(HBSS),10倍(西格玛,目录号:H1641-500ML)
    11. 碳酸氢钠(NaHCO 3 )(Sigma,目录号:S5761-500G)
    12. L-半胱氨酸盐酸盐一水合物(半胱氨酸-HCl)(Sigma,目录号:C7880-100G)
    13. 木瓜蛋白酶(Worthington Biochemical Corp,目录号:LS003126)
    14. 细胞培养级无菌水(Hyclone,目录号:SH30529.03)
    15. 牛血清白蛋白(BSA),级分V(Fisher,目录号:BP1600-100)
    16. 大豆胰蛋白酶抑制剂(Sigma,目录号:T6522-1G)
    17. 磷酸盐缓冲盐水(PBS),10x溶液(Fisher,目录号:SH3037803)
    18. 1x PBS溶液(使用细胞培养级无菌水从10x PBS稀释)
    19. 多聚甲醛(PFA)(Fisher,目录号:AC416780030)
    20. HBSS解剖培养基(4°C储存长达3个月)(见食谱)
    21. 解离介质(在4°C下储存长达3个月)(见食谱)
    22. L-半胱氨酸50x库存(见食谱)
    23. 木瓜蛋白酶解离溶液和抑制剂解决方案用于分离原代大鼠皮层神经元(使用当天新鲜)(见食谱)
      1. 酶解决方案
      2. 重抑制剂解决方案
      3. 光抑制剂溶液
    24. 4%PFA溶液(见食谱)

  4. 电镀和培养基
    1. Neurobasal Media(Gibco,目录号:21103-049)
    2. 胎牛血清,热灭活(Hyclone,目录号:SH30070.03HI)
    3. B27 Media Supplement,50x(Gibco,目录号:17504-044)
    4. Glutamax(Gibco,目录号:35050-061)
    5. 青霉素/链霉素抗生素溶液(Gibco,目录号:15140-122)
    6. 用于播种的皮质神经元培养基(在4°C下储存最多10天)(参见食谱)
    7. 用于喂养的皮质神经元培养基(在4°C下储存最多10天)(参见食谱)

  5. 微流体装置的制备,培养和再利用
    1. Xona Microfluidics ®微通道培养微流体装置
      1. 450μm微槽屏障标准神经元设备(Xona Microfluidics,目录号:SND450)
      2. 900μm微槽屏障标准神经元装置(Xona Microfluidics,目录号:SND900)
    2. 康宁盖玻片盖玻片(康宁,目录号:2975-244)
    3. 聚-D-赖氨酸氢溴酸盐,MW 70,000-150,000(Sigma,目录号:P0899-50MG)
    4. 硼酸(Fisher,目录号:S79802)
    5. Borax,无水(MP Biomedicals,目录号:190309)
    6. HCl,12 N(Fisher,目录号:A144-500)
    7. 细胞培养级无菌水(Hyclone,目录号:SH30529.03)
    8. RBS清洁剂溶液(Thermo,目录号:27950)
    9. 100%乙醇(Decon Labs,目录号:2705HC)
    10. 0.1 M硼酸盐缓冲溶液(见食谱)
    11. Poly-D-Lysine 2.5 mg / ml储备液(见食谱)
    12. Poly-D-Lysine100μg/ ml涂层溶液(参见配方)

  6. 鱼藤酮治疗
    1. DMSO(Sigma,目录号:D2438)
    2. Rotenone(Sigma,目录号:R8875-1G)
    3. Rotenone股票解决方案(见食谱)
    4. Rotenone 2x和1x解决方案(参见食谱)
    5. DMSO车辆控制2x和1x解决方案(参见食谱)

设备

  1. CO 2 坦克&nbsp;
  2. CO 2 单级流量计调节器(Western Medica,型号:M1-320-12FM)
  3. CO 2 气体麻醉箱(Braintree Scientific,型号:AB-2)
  4. Motic Dissection显微镜(Motic,型号:SMZ-168)
  5. Roboz手术器械解剖工具&nbsp;
    1. Dumont#5镊子,直尖(Roboz,目录号:RS-5060)
    2. 杜蒙#5镊子,45度尖(Roboz,目录号:RS-5005)
    3. Straight,Sharp-Blunt操作剪刀(Roboz,目录号:RS-6812)
    4. 超细微解剖剪刀(Roboz,目录号:RS-5882)
    5. Micro Dissecting Spring Scissors(Roboz,产品目录号:RS-5603)
  6. Sorvall Legend T台式离心机(Thermo,型号:75004367)
  7. Nuaire Class II A2型层流无菌培养罩,带真空垃圾系统(Nuaire,型号:NU-425-400)
  8. 徕卡Fiberlight光源(徕卡,型号:EEG2823)
  9. 奥林巴斯光学显微镜(奥林巴斯,型号:CKX31)
  10. HeraCell CO 2 - 和温控培养箱(Thermo,型号:51022394)
  11. Isotemp 110加热水浴(Fisher Scientific,型号:15-460-10)
  12. Wheaton玻璃载玻片染色罐(Fisher Scientific,型号:22-309-247)
  13. 一次性转移移液器(Fisher Scientific,目录号:13-711-20)
  14. Rocking Shaker Plate(Labnet,型号:S-2035)
  15. 加热搅拌板(康宁,型号:PC-420)
  16. Accumet碱性pH计(Fisher Scientific)
  17. 涡旋混合器(Labnet,型号:VX100)
  18. 规模(丹佛仪器,型号:APX-100)
  19. Millipore Milli-Q超纯水系统
  20. 各种机械移液器和相应的无菌吸头
  21. 血细胞计数器和细胞计数器
  22. 温度计
  23. 高压灭菌器
  24. 化学通风柜
  25. 4°C冰箱
  26. -20°C冰柜
  27. -80°C冰柜

程序

  1. 提前准备解剖和电镀
    1. 根据下面的食谱准备硼酸盐缓冲溶液,解离介质和HBSS解剖介质。
    2. 根据以下食谱准备必要数量的播种培养基和饲喂培养基。准备的数量取决于计划实验的规模。
      1. 每个完整的神经元制剂需要大约25毫升的种子培养基,每个装置需要大约5毫升的喂养培养基用于电镀和维护。
      2. 制备的培养基可以保存长达10天,并可用于随后的神经元制备。因此,为后续使用制作足够的媒体是合理的。&nbsp;

  2. 在解剖和电镀前准备2天
    1. 解冻Poly-D-Lysine
      1. 从-80℃下取必要数量的2.5mg / ml聚-D-赖氨酸储备溶液(400μl)的等分试样。
      2. 置于冰上,在4°C下解冻过夜。
    2. 酸洗和消毒盖玻片
      1. 在无菌培养罩中,将所需数量的盖玻片(每个待镀设备1个,加上2个额外的)放入载玻片染色罐中。
      2. 加入50毫升1N HCl(在无菌细胞培养水中制备)。&nbsp;
      3. 将盖子放在染色罐上,然后将罐子放在罩子外面的振动筛板上。在振动筛板上轻轻搅拌盖玻片1小时。
      4. 将罐子放回无菌培养罩中,通过移液管或抽吸从罐中取出HCl。
      5. 将无菌细胞培养水加入罐中,盖上盖子,然后将罐子放在罩子外面的振动筛板上。重复此水冲洗3 x 10分钟。
      6. 通过移液管或抽吸除去最后的水洗液,并加入50ml 70%乙醇溶液以对盖玻片进行灭菌。盖上广口瓶,在振动筛板上轻轻搅拌30分钟。
      7. 通过移液管或抽吸除去乙醇洗液,并向盖玻片中加入50ml无菌培养水。盖上广口瓶,在振动筛板上轻轻搅拌10分钟。
      8. 在无菌培养罩中,从染色罐中取出盖玻片,并在敞开的10cm培养皿中静置以风干。将盖玻片的边缘向上推到培养皿的侧面 - 不要让盖玻片平放。
      9. 干燥后,将盖玻片暴露在无菌培养罩的紫外线(UV)下20分钟,对盖玻片进行灭菌。
      10. 紫外线照射后,将盖玻片放入培养皿内并盖上盖子。在密闭容器中或在无紫外线照射的无菌培养罩中储存过夜。

  3. 在解剖和电镀前准备1天
    1. 涂有Poly-D-Lysine的盖玻片
      1. 在无菌培养罩中进行该程序。
      2. 安排盖玻片,使它们平放,不重叠,放在10厘米培养皿的底部。
      3. 通过将2.5mg / ml聚-D-赖氨酸储备溶液以400μl,2.5mg / ml的比例添加到无菌0.1M硼酸盐缓冲液中,每10cm培养皿制备6ml100μg/ ml聚-D-赖氨酸涂层溶液。聚-D-赖氨酸原液至9.6毫升,0.1M硼酸盐缓冲液。
      4. 混合并无菌过滤100μg/ ml聚-D-赖氨酸涂层溶液。
      5. 用100μg/ ml聚-D-赖氨酸涂层溶液覆盖盖玻片,将盖子放在培养皿上,让溶液在4°C下盖上盖玻片过夜。
    2. 消毒微流体装置
      1. 在无菌培养罩中工作,从包装中取出设备。
      2. 将装置槽侧向上(即,倒置)放入无菌的10cm培养皿中。
      3. 将设备浸入100%乙醇中30分钟。
      4. 除去乙醇,将设备浸入70%乙醇中30分钟。
      5. 从乙醇中取出装置,并浸入一盘无菌细胞培养水中。
      6. 在无菌培养皿的边缘上以一定角度设置装置,并允许在无菌培养罩中完全干燥。
      7. 放在培养皿中并盖上盖子。在密闭容器中或在无菌培养罩中在黑暗中储存过夜,无需紫外线或荧光灯照射。
      8. 不要随时将设备暴露在紫外线下。

  4. 解剖日
    1. 根据下面的食谱准备木瓜蛋白酶溶液和抑制剂溶液。
    2. 将饲喂培养基,种子培养基,木瓜蛋白酶消化液和木瓜蛋白酶抑制剂溶液置于37°C水浴中。
    3. 将HBSS解剖溶液置于冰上。将约150ml HBSS等分至待用于工作台,并将25ml HBSS保持无菌以用于罩中。
    4. 冲洗干燥盖玻片
      1. 在无菌培养罩中工作,使用无菌镊子从聚-D-赖氨酸溶液中取出盖玻片,并将角度放置在无菌培养皿的边缘上以使其干燥。
      2. 干燥后,将每个盖玻片3 x浸入无菌培养水中冲洗,让水在蘸水之间滴落。
      3. 将盖玻片浸入无菌1x PBS中,让溶液滴下,然后返回培养皿,支撑在培养皿边缘,使其完全干燥。不要暴露在紫外线下。
    5. 将微流体设备连接到盖玻片上并使用喂养介质进行灌注 - 参见图1,了解设备的放置和方向
      1. 在无菌培养罩中进行该程序。
      2. 使用无菌镊子,将单个Poly-D-Lysine涂层的盖玻片放在无菌的6 cm培养皿的底部,确保将Poly-D-Lysine涂层面朝上。
      3. 使用第二组无菌镊子,提起微流体装置并将其从微槽侧向下放置到盖玻片的中心。 (图1A)
      4. 使用镊子的钝端将设备轻轻按压到盖玻片上,确保轻轻挤出任何气泡以确保密封。
      5. 将盖子放在培养皿上以保持无菌状态,并在光学显微镜下检查设备组件,以确保微槽和饲养池周围的紧密密封。&nbsp;
      6. 返回无菌培养罩并用温热的喂养培养基灌注装置如下(图1B和1C):
        1. 在左上方的水库中加入100μl温热的喂养培养基。注意确保介质进入并填充连接两个左侧储存器的通道。
        2. 等待2分钟以确保介质填充轴突通道和微槽。
        3. 接下来,在右上方的水库中加入100μl温热的培养基。
        4. 等待2分钟以确保介质填充了正常通道。
        5. 向左下和右下储液器各添加100μl温热介质。
        6. 将盖子放在固定装置的培养皿上,放置在细胞培养箱(37°C,5%CO 2 )中至少1小时,然后铺设神经元。


      图1.使用培养基安装,定向和引发微流体装置的示意图。 A.将微流体装置放置在盖玻片上。 B.设备功能和方向。 C.添加到主要设备的媒体顺序。

    6. 原代皮层神经元解剖和细胞制备
      在替补席上
      1. 按照已建立的实验室方法并根据符合匹兹堡大学实验动物研究部(DLAR)和机构动物护理要求的批准动物方案,人道地处死E18怀孕雌性大鼠并解剖胚胎大鼠原代皮质(脑膜移除)。和使用委员会(IACUC)。这些要求因机构而异。我们的方法改编自Ghosh和Greenberg(1995)描述的先前工作,并且如我们实验室先前所述(Arnold et al。,2011)。细胞产量变化,每个胚胎约0.5-1.0×10 6个细胞。
      2. 轻轻地将所有皮质转移到含有9ml HBSS解剖培养基的冰上的15ml锥形管中。
      3. 解剖完成后,移至无菌培养罩继续手术。

      在无菌培养罩中
      1. 使用移液管轻轻移除皮质中的HBSS并丢弃HBSS。
      2. 每次洗涤使用5 ml冷HBSS轻轻洗涤皮质3次。每次洗涤后,使用移液管轻轻从皮质中去除HBSS并丢弃。
      3. 在最后一次HBSS洗涤后,立即向皮质中加入5ml预热的酶溶液,并在37℃下孵育20分钟。
      4. 使用移液管移除酶溶液,立即将预热的光抑制剂溶液(10 ml)加入皮质中1分钟。
      5. 使用移液管移除光抑制剂溶液,立即将重抑制剂溶液(5 ml)加入皮质中,在37°C下保持2分钟。
      6. 使用移液管移除重抑制剂溶液,每次洗涤使用5 ml 37°C接种培养基轻轻洗涤皮质3次。每次洗涤后,使用移液管轻轻地从皮质中取出培养基并丢弃。
      7. 将新鲜的5ml种子培养基加入皮质中,使用P1000(1 ml)机械移液管,上下移液约20次,将组织和细胞团块分解成均质细胞悬液。如果细胞仍处于团块状态,您可能需要更长时间(5-10次)研磨,但 不要过度研磨 。 细胞悬液应看起来混浊,并且不应留下明显的大颗粒或细胞团块。或者,在20次研磨后,细胞可以通过40μm无菌细胞过滤器筛。
      8. 统计细胞
        1. 将10μl等分试样的悬浮细胞与90μl种子培养基混合。
        2. 使用血细胞计数器计数细胞并计算悬浮液中的细胞/ ml。

  5. 电镀微流体装置
    对于具有450μm或900μm宽度微槽屏障(Xona微流体)的标准2室微流体神经元装置,将初级皮层神经元以每个装置50,000个细胞铺板。为了确保电镀不会导致细胞被迫通过微槽进入设备的轴突,非细胞侧,请按照以下步骤进行(图2):
    1. 在种子培养基中将细胞悬浮液调节至2.5×10 6个/ ml细胞/ ml。每个待镀设备准备足够的20μl细胞溶液。搁置。
    2. 从培养箱中取出固定有微流体装置的培养皿,并将它们放入无菌培养罩中。
    3. 从所有储液器中取出用于灌注设备的所有介质。
    4. 向每个装置的左上方储库(轴突侧)添加100μl进料培养基。
    5. 向连接的左下方储液器(轴突侧)添加100μl进料培养基。等待1分钟使培养基在装置的轴突侧平衡。 (见注释)
    6. 在装置的最佳侧面,向右上方的水库(躯体侧)添加10μl细胞悬浮液。
      注意:将10μl细胞悬液置于储液器连接通道的连接处(见图2)。
    7. 盖住固定装置的培养皿,并在光学显微镜下检查以确保细胞进入右上和右下贮存器之间的通道。 (见注释)
    8. 1分钟后,将另外10μl细胞悬浮液加入连接的右下方储液器(躯体侧)。
    9. 将盖子放在固定装置的培养皿上,然后将其放回培养箱中。让细胞附着10分钟。
    10. 10分钟后,将设备从培养箱中取出并返回无菌培养罩。
    11. 轻轻地在左上方的水库(轴突侧)添加额外的100μl喂养培养基。
    12. 轻轻地在连接的左下方储液器(轴突侧)添加额外的100μl进料培养基。
    13. 在步骤E12之后,立即轻轻地将200μl喂养培养基添加到细胞侧右上方的储库(躯体侧)。&nbsp;
    14. 紧接着,轻轻地将200μl喂养培养基添加到连接的右下方水库(躯体侧)。
      注意:
      1. 添加媒体时要缓慢而温和;强力或过快地添加培养基会将细胞从通道中洗掉。
      2. 现在左边的每个水库都有200μl的饲喂培养基,右边的每个水库都有210μl的饲养培养基。
    15. 将盖子放在固定设备的6厘米培养皿上。&nbsp;
    16. 将覆盖的6cm培养皿保持装置放入10cm培养皿中。最多三个6厘米的菜肴将放入一个10厘米的菜肴中。将盖子放在10厘米培养皿上,然后将设备放回培养箱中。这种盘碟系统用于在培养期间帮助减少蒸发并保持设备中的介质水平。


      图2.将神经元植入微流体装置的示意图

  6. 喂养维持和鱼藤酮治疗
    1. 维护细胞
      1. 接种后,每3-4天在每个装置的四个贮存器中加入10μl喂养培养基,以维持培养基水平。将温热的培养基添加到无菌培养罩中的装置中。
      2. 在初始治疗之前不会发生媒体变化。
    2. 慢性鱼藤酮和DMSO载体治疗
      在体外日 7(DIV7),通过在轴突侧和躯体侧储库中的1/2介质变化添加鱼藤酮或DMSO载体对照的初始处理,如下:&nbsp;
      1. 在温热的喂养培养基中制备2倍浓度的所需最终鱼藤酮浓度(参见用于溶液制备的配方)。
      2. 同时,使用相应的2x浓度的DMSO作为载体对照制备温热的喂养培养基(参见用于溶液制备的配方)。
      3. 从培养箱中取出保持微流体装置的培养皿。
      4. 在无菌培养罩中,从每个装置上的四个贮存器中的每一个中移除100μl培养基。
      5. 对于每个装置,将100μl2x鱼藤酮或2x DMSO溶液添加到四个储库中的每一个中。
      6. 在整个装置中,最终浓度为1x鱼藤酮或1x DMSO。
    3. 治疗持续1-2周,每3-4天使用1x浓度培养基更换½培养基。
      1. 在温热的饲喂培养基中制备1x浓度的所需最终鱼藤酮浓度和1x浓度的DMSO载体对照(参见用于溶液制备的配方)。
      2. 如上所述,对每个储液器重复½介质更换。

  7. 修复神经元(图3)
    1. 在实验结束时,从所有储存器中移除介质。
    2. 立即在左上和右上储液池中加入100μl温热的1x PBS。
    3. 让PBS流到底部储液器1分钟。
    4. 立即取出所有PBS。
    5. 向每个顶部储液器中加入100μl温热的4%PFA。
    6. 让细胞在室温下固定20分钟。
    7. 删除所有PFA,并妥善处理。&nbsp;
    8. 向每个顶部储库中加入200μlPBS。
    9. 冲洗5分钟。
    10. 去除PBS冲洗液。
    11. 重复PBS冲洗2次。
    12. 用PBS填充每个储库。
    13. 盖上装置,储存在4°C直至用于进一步分析。&nbsp;


    图3.固定微流体装置中神经元的示意图

  8. 重复使用微流体装置
    如果培养物在实验性处理开始之前死亡,则装置没有适当地安装并且细胞进入装置的非细胞体侧,或者培养物不能用于实验,我们发现该装置可以重复使用成功。不要重复使用经过实验性处理的设备,如药物或有毒物质(见注释)。从安装有活性培养物的微流体装置开始,按照以下步骤重复使用设备:
    1. 使用移液器从设备容器中取出介质。&nbsp;
    2. 将手指放在盖玻片的角上,然后使用镊子轻轻地抓住设备的边缘,并轻轻向上拉一定角度,直到设备没有盖玻片。
    3. 用蒸馏水轻轻冲洗设备,然后将槽边朝上放入培养皿中。
    4. 将设备浸入1x RBS清洁剂溶液中。盖上培养皿,在室温下在摇床上轻轻搅拌过夜。
    5. 取出RBS清洁剂,用培养级水3次洗涤装置1小时,在室温下在摇床上轻轻搅拌。
    6. 最后一次洗涤后,将设备浸没在水中,在光学显微镜下检查设备,以确认所有细胞碎片已从储存器和凹槽中移除。
    7. 取下最后一次清洗,然后在无菌培养罩中进行剩余的步骤。
    8. 将装置(槽侧朝上)转移到无菌培养皿中,然后将装置浸没在无菌培养级水中。盖上培养皿,在室温下在摇床上轻轻搅拌过夜。
    9. 在无菌培养罩中,将装置从无菌洗涤液中取出,并在无菌培养皿中风干以储存以备将来使用,或者执行上述步骤C2中列出的灭菌程序以立即使用。

数据分析

在用于实验,数据采集和数据分析之前,请仔细检查设备和培养质量。在处理之前细胞出现明显窘迫的装置培养物不应用于实验或进一步分析。尽管设备的设计使得细胞不能穿过微通道,但是一些细胞偶尔会穿过微通道。特别是Glia能够容易地穿过。偶尔的神经元体细胞不是一个问题。如果经过一周的培养,您会注意到轴突侧有大量的神经元胞体,停止使用这种器械培养物。如果您在实验后发现轴突侧有许多somata,请不要使用此设备文化进行数据分析。轴突侧的大量somata可能表明电镀有问题(见注释)或装置没有正确连接到盖玻片上。

笔记

在微流体装置中电镀神经元(程序E)

  1. 细胞的铺板是维持体细胞和轴突之间隔离的关键步骤。确保装置的轴突室侧的储液器和通道在装置的正常隔室侧向储液器中添加细胞之前1-2分钟充满喂养介质。如果在添加细胞时轴突隔室是空的,则流体动力学将迫使分离的细胞通过微通道并进入轴突通道空间。如果发生这种情况,该装置及其神经元不能用于进一步的实验,因为轴突将不再是孤立的群体。 (步骤E2-E3)
  2. 在步骤E4-E6中,当将细胞添加到装置的正常侧时,建议始终通过显微镜检查细胞是否进入正常的隔室侧通道。流体动力学应将细胞拉入通道。如果您没有观察到这一点,请返回无菌培养罩,尝试用移液管轻轻取出细胞。确保从右上方和右下方的水库中移除所有介质,并再次尝试将细胞放置在右上方的水库中,水库与水道连接。

重复使用微流体装置(程序H)
  1. 只有重复使用不的设备才能暴露于培养基中不常见的任何外源性处理,如药物,毒物(如鱼藤酮)或组织固定剂如PFA。如果在治疗实验期间已经使用过该装置,或者如果细胞和装置已暴露于固定剂,则该装置不能重复使用。 因此,完成实验的任何设备都无法重复用于其他实验。
  2. 该再利用方法仅适用于压在细胞外基质涂覆的盖玻片上的微流体装置。如果使用等离子键合技术将器件绑定到盖玻片上(参见制造商的说明),则不能重复使用。
  3. 在我们的研究中,我们发现来自新设备和重复使用设备的培养物没有表现出任何可辨别的差异。此外,我们观察到在重新使用的设备中完成的新设备和实验中完成的实验所获得的结果没有差异。

食谱

  1. 0.1 M硼酸盐缓冲溶液
    1. 在400 ml细胞培养级无菌H 2 O中加入1.24 g硼酸和1.002 g无水硼砂
    2. 盖上并在室温下在搅拌板上搅拌过夜
    3. 将pH调节至8.4
    4. 在无菌培养罩中,过滤灭菌到高压灭菌的培养基瓶中
    5. 在4°C下储存长达3个月
  2. 聚-D-赖氨酸2.5 mg / ml储备液
    1. 将冷冻的冻干粉末在4℃下在冰上解冻过夜
    2. 在无菌培养罩中准备库存
    3. 向50mg聚-D-赖氨酸中加入20ml 0.1M无菌硼酸盐缓冲液
    4. 在无菌培养罩中,使用10ml注射器和0.22μmPES注射器过滤器对储备溶液进行过滤灭菌
    5. 将400μl原液分装到无菌微量离心管中
    6. 储存在-80°C
  3. 聚-D-赖氨酸100μg/ ml涂层溶液
    1. 在4°C冰上解冻一份Poly-D-Lysine储备液(400μl,2.5 mg / ml)过夜
    2. 在无菌培养罩中,加入9.5ml无菌0.1M硼酸盐缓冲液
    3. 最终浓度:100μg/ ml聚-D-赖氨酸
  4. 用于播种的皮质神经元培养基
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:400px; height:00px;” border =“0”cellspacing =“2”cellpadding =“6”>试剂
    %vol / vol
    500 ml
    Neurobasal媒体
    91.5%
    457.5毫升
    B27补充
    2%
    10毫升
    Glutamax 100x
    1%
    5毫升
    笔/ Strep
    0.5%
    2.5毫升
    FBS
    5%
    25毫升 注意:在无菌培养罩中准备所有溶液。&nbsp;
    1. 将所有试剂混合在无菌培养罩中,过滤灭菌到高压灭菌的培养瓶中
    2. 在4°C下储存长达10天
  5. 用于喂养的皮质神经元培养基
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:400px; height:100px;” border =“0”cellspacing =“2”cellpadding =“6”>试剂
    %vol / vol
    500 ml
    Neurobasal媒体
    96.5%
    457.5毫升
    B27补充
    2%
    10毫升
    Glutamax 100x
    1%
    5毫升
    笔/ Strep
    0.5%
    2.5毫升
    注意:在无菌培养罩中准备所有溶液。
    1. 将试剂在无菌培养罩中混合,过滤灭菌到高压灭菌的培养基瓶中
    2. 在4°C下储存长达10天
  6. HBSS解剖媒体
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:400px; height:100px;” border =“0”cellspacing =“2”cellpadding =“6”>试剂
    的 金额。
    最终结论
    10倍HBSS
    100毫升
    1x
    的NaHCO <子> 3 &NBSP; 350毫克
    4.2 mM
    HEPES,1 M
    10毫升
    10 mM
    葡萄糖
    4.3克
    35 mM
    用细胞培养级无菌H 2 O进行1L 注意:
    1. 不要调节pH值。
    2. 在无菌培养罩中准备所有溶液。
    1. 将试剂在无菌培养罩中混合,过滤灭菌到高压灭菌的培养基瓶中。
    2. 在4°C下储存长达3个月
  7. 解离媒体 class =“ke-zeroborder”bordercolor =“#000000”style =“width:400px; height:100px;” border =“0”cellspacing =“2”cellpadding =“4”>试剂
    的 金额。
    Na 2 SO 4 ,1M
    20.44毫升
    K 2 SO 4 ,0.5M
    15毫升
    MgCl 2 ,1M 1.45毫升
    CaCl 2 ,100 mM
    0.625毫升
    HEPES,1 M
    250μl
    葡萄糖,1 M
    5毫升
    酚红,0.5%
    0.5毫升
    NaOH,0.1N
    0.5毫升
    用细胞培养级无菌H 2 O进行250ml 注意:
    1. 不要调节pH值。
    2. 在无菌培养罩中准备所有溶液。
    1. 将试剂在无菌培养罩中混合,过滤灭菌到高压灭菌的培养基瓶中
    2. 在4°C下储存长达3个月
  8. L-Cysteine 50x库存
    1. 称取160毫克,然后在无菌培养罩中制备
    2. 溶于10ml无菌解离培养基中
    3. 将样品置于225μl的无菌微量离心管中,并在-80℃下储存
  9. 木瓜蛋白酶解离溶液和抑制剂溶液分离原代大鼠皮层神经元
    注意:
    1. 在使用当天使这些解决方案变得新鲜。
    2. 首先将解离培养基分装到无菌培养罩中的无菌15 ml锥形管中,用于下面的每种溶液。
    3. 使用pH计在工作台上准备溶液。
    4. 制备溶液后,返回无菌培养罩。
    5. 对于每种溶液,使用10 ml无菌注射器和注射器过滤器将溶液无菌过滤到新的无菌15 ml锥形管中。
    6. 在制备和最终pH调节后,使用10 ml注射器和0.22μmPES注射器过滤器,在无菌培养罩下将所有溶液过滤灭菌到新的无菌15 ml锥形瓶中。
    1. 酶解决方案
      10毫升解离介质
      0.2毫升50倍半胱氨酸-HCl
      200 U木瓜蛋白酶溶液
      脉冲在涡旋混合器上
      用0.1N NaOH调节pH至7.4
    2. 重抑制剂解决方案
      6毫升解离介质
      将60 mg BSA(4°C)加入6 ml DM中 60毫克大豆胰蛋白酶抑制剂(4°C)
      在涡旋混合器上脉动,然后放在振荡器上混合直至完全溶解在溶液中 用0.1N NaOH调节pH至7.4
    3. 光抑制剂解决方案
      9毫升解离介质
      加入1毫升重抑制剂溶液
      脉冲在涡旋混合器上
      用0.1N NaOH调节pH至7.4
  10. Rotenone股票解决方案
    1. 使用新鲜鱼藤酮和DMSO制备原料
    2. 在DMSO中制备5 mM鱼藤酮&nbsp;
    3. 等分成100μl等分试样并储存在-20℃
    4. 为所有实验准备足够的库存
  11. Rotenone 2x和1x解决方案
    1. 使用当天,使用前立即新鲜
    2. 准备无菌培养罩
    3. 在室温下解冻等份的5mM鱼藤酮,避光
    4. 直接稀释至神经元喂养培养基至第一次治疗的最终所需治疗浓度的2倍,并用于后续治疗的1倍
  12. DMSO车辆控制2x和1x解决方案
    1. 使用当天,使用前立即新鲜
    2. 准备无菌培养罩
    3. 将DMSO直接稀释到神经元喂养培养基至第一次处理的最终所需处理浓度的2倍,并用于后续处理的1倍
    4. 应制备载体对照培养基以具有与鱼藤酮处理培养基中相同的DMSO终浓度
  13. 4%PFA解决方案
    准备2升4%PFA
    200毫升10倍PBS
    80克多聚甲醛
    Milli-Q超纯水至2升
    1. 准备化学通风橱&nbsp;
    2. 将1L超纯水加入带有大搅拌棒的2L烧瓶中
    3. 加入112.5μl的10N NaOH
      注意:
      &nbsp; &NBSP; &NBSP; &NBSP;我。多聚甲醛在碱中比在酸中溶解更好。
      &nbsp; &NBSP; &NBSP; &NBSP;ⅱ。在此阶段省去PBS以消除缓冲。
    4. 将温度计悬浮在溶液中并将烧瓶置于加热的搅拌板上并搅拌
    5. 将溶液置于低于60°C并继续搅拌直至PFA溶解 注意:密切监控;不要让多聚甲醛溶液超过60°C。
    6. 当PFA溶解时,用pH试纸将pH值调至7.4 注意:请勿使用pH计。根据需要用HCl和NaOH调节pH。
    7. 加入200毫升10倍PBS
    8. 用超纯水将溶液加入2L
    9. 用pH试纸重新检查pH值,确认最终pH值为7.4
    10. 过滤灭菌并在50ml锥形管中制备40ml等分试样
    11. 储存于-20°C直至使用

致谢

这项工作得到了NIH(R01NS077954,S.B.B。)和匹兹堡大学医学院学术基金会研究基金(S.B.B)的资助。
微流体装置电镀和培养方法改编自Park 等人(2006)所述的先前工作。
如Arnold et al。(2011)所述,我们之前的工作对慢性低剂量鱼藤酮暴露治疗方法进行了修改。

利益争夺

作者没有披露利益冲突。

伦理

所有动物程序均经匹兹堡大学的机构动物护理和使用委员会批准,并符合美国国立卫生研究院在实验动物护理和使用指南中提出的指导。所有安乐死方法都与最新的美国兽医协会(AVMA)动物安乐死指南一致。

参考

  1. Arnold,B.,Cassady,S.J。,VanLaar,V。S. and Berman,S。B.(2011)。 整合神经元中线粒体动力学的多个方面:慢性鱼藤酮模型中与年龄相关的差异和动态变化。 Neurobiol Dis 41(1):189-200。
  2. Ghosh,A。和Greenberg,M。E.(1995)。 神经元中的钙信号传导:分子机制和细胞结果。 Science 268(5208):239-247。
  3. Millet,L。J.和Gillette,M。U.(2012)。 超过一个世纪的神经元培养:从悬滴到微流体装置。 Yale J Biol Med 85(4):501-521。
  4. Park,J.W.,Vahidi,B.,Taylor,A.M.,Rhee,S.W。和Jeon,N.L。(2006)。 用于神经科学研究的微流体培养平台。 Nat Protoc 1 (4):2128-2136。
  5. Taylor,A.M.,Blurton-Jones,M.,Rhee,S.W.,Cribbs,D.H.,Cotman,C.W。和Jeon,N.L。(2005)。 用于CNS轴突损伤,再生和转运的微流体培养平台。 Nat方法 2(8):599-605。
  6. Taylor,A.M.,Menon,S。和Gupton,S.L。(2015)。 被动微流体室,用于长期成像轴突引导以响应可溶性梯度。 Lab Chip 15(13):2781-2789。
  7. Taylor,A.M.,Rhee,S.W.,Tu,C.H.,Cribbs,D.H.,Cotman,C.W。和Jeon,N.L。(2003)。 用于神经科学研究的微流体多室设备。 Langmuir 19( 5):1551-1556。
  8. Van Laar,V。S.和Berman,S。B.(2013)。 神经元线粒体动力学和生物能量学的相互作用:对帕金森病的影响。 Neurobiol Dis 51:43-55。
  9. Van Laar,V.S。,Arnold,B.,Howlett,E.H.,Calderon,M.J.,St Croix,C.M.,Greenamyre,J.T。,Sanders,L.H。和Berman,S.B。(2018)。 分区轴突线粒体生物发生的证据:远端轴突线粒体DNA复制增加,作为对帕金森病的早期反应 - 相关的压力。 J Neurosci 38(34):7505-7515。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Van Laar, V. S., Arnold, B. and Berman, S. B. (2019). Primary Embryonic Rat Cortical Neuronal Culture and Chronic Rotenone Treatment in Microfluidic Culture Devices. Bio-protocol 9(6): e3192. DOI: 10.21769/BioProtoc.3192.
  2. Van Laar, V. S., Arnold, B., Howlett, E. H., Calderon, M. J., St Croix, C. M., Greenamyre, J. T., Sanders, L. H. and Berman, S. B. (2018). Evidence for compartmentalized axonal mitochondrial biogenesis: mitochondrial DNA replication increases in distal axons as an early response to Parkinson's disease-relevant stress. J Neurosci 38(34): 7505-7515.
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