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Nov 2019

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Co-culture of Murine Neurons Using a Microfluidic Device for The Study of Tau Misfolding Propagation
利用微流体装置共培养小鼠神经元研究Tau的错误折叠传播   

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Abstract

The deposition of misfolded, aggregated tau protein is a hallmark of several neurodegenerative diseases, collectively termed “tauopathies”. Tau pathology spreads throughout the brain along connected pathways in a prion-like manner. The process of tau pathology propagation across circuits is a focus of intense research and has been investigated in vivo in human post-mortem brain and in mouse models of the diseases, in vitro in diverse cellular systems including primary neurons, and in cell free assays using purified recombinant tau protein. Here we describe a protocol that takes advantage of a minimalistic neuronal circuit arrayed within a microfluidic device to follow the propagation of tau misfolding from a presynaptic to a postsynaptic neuron. This assay allows high-resolution imaging as well as individual manipulation of the releasing and receiving neuron, and is therefore beneficial for investigating the propagation of tau and other misfolded proteins in vitro.

Keywords: Protein misfolding propagation (蛋白质错误折叠传播), Microfluidic device (微流体装置), Hippocampal culture (海马体培养), Tau (Tau), Imaging (成像), Image analysis (图像分析)

Background

The propagation of misfolded protein throughout the brain underlies the spread of pathology in several neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s and Prion disease (Goedert et al., 2010; Davis et al., 2018; Hallinan et al., 2019a). Understanding the underlying mechanisms may help limit disease progression and is therefore an area of intense research. Several in vivo and in vitro assays exist that monitor this process, including mouse models that develop neurodegeneration in response to overexpressing mutant human tau (Allen et al., 2002; Ramsden et al., 2005; SantaCruz et al., 2005; Gibbons et al., 2017). In vitro models include cell lines expressing biosensors that monitor aggregation (Kfoury et al., 2012; Holmes et al., 2014) and primary neuronal cultures, including those in microfluidic devices (Wu et al., 2013 and 2016, Takeda et al., 2015). In this protocol we describe in detail an in vitro assay to monitor the seeding and propagation of tau misfolding across connected neurons. This protocol builds on previously established assays, as described above, but additionally allows to monitor not just the passage of misfolded tau from one neuron to the next, but also the seeding in the previously unaffected cell, thus fully recapitulating inter-neuronal propagation. The microfluidic setup further allows individual manipulation of the pre- and postsynaptic cell, and high resolution live and fixed cell imaging of both connected neurons. This assay can be adapted for the study of the spread of other proteinopathies, such as prion or α-synuclein pathology. In addition, the basic directionalised and connected setup allows the culture of a range of different neuronal subtypes and thus the study of various other trafficking- and spread-related neurotoxic agents, including that of viruses and environmental insults such as nanoparticle toxicity.

Materials and Reagents

  1. Microfluidic device preparation
    1. Microfluidic devices
      Note: Microfluidic devices are commercially available from Xona Microfluidics, or can be manufactured as previously described in Taylor et al., 2005 and Holloway et al., 2019. For unidirectional outgrowth we recommend using the “arrow” devices described in Holloway et al., 2019.
    2. 22 x 50 mm rectangle coverslips (0.16-0.19 mm thickness, Smith Scientific, catalog number: NPS16/2250 )
    3. 94/16MM Petri dishes, non-vented (Greiner Bio-one, catalog number : 632180 )
    4. 3 M Scotch-tape
    5. poly-D-Lysine hydrobromide (PDL, Sigma, catalog number: P7886 )
    6. Sterile ddH2O
    7. 70% Ethanol (EtOH)
    8. Neurobasal medium (Gibco, catalog number: 21103-049 )

  2. Primary neuron culture and transfection
    1. 15 ml Falcon Conical Tubes 17/120MM (Cellstar, catalog number: 188271 )
    2. 50 ml Falcon Conical Tube 30/115 (Cellstar, catalog number: 227261 )
    3. 40 µm sterile cell strainer (Fisher Scientific, catalog number: 15360801 )
    4. Timed-pregnant female mouse, E15-E18
      Note: We routinely use C57Bl/6 mice.
    5. Glutamax supplement (Gibco, catalog number: 35050-038 )
    6. Neurobasal Media (NBM) (Gibco, catalog number: 21103-049 )
    7. Fetal Bovine Serum (FBS) (Gibco, catalog number: 10270-106 )
    8. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, catalog number: 41965-039 )
    9. Opti-MEM reduced serum medium with glutamax supplement (Gibco, catalog number: 51985-026 )
    10. DNase (Sigma, catalog number: DN25 ; optional)
    11. B27 Media Supplement, 50x (Gibco, catalog number: 17504-044 )
    12. Lipofectamine2000 (Gibco, catalog number: 1168027 )
    13. 0.5% Trypsin-EDTA (Gibco, catalog number: 15400-054 )
    14. Phosphate Buffered Saline (DPBS), no Calcium (Ca2+) and Magnesium (Mg2+) (Gibco, catalog number: 14190-094 )
    15. PDL solution (see Recipes)
    16. Culture medium (complete NBM) (see Recipes)
    17. DNase solution (see Recipes)

  3. Fixation and imaging
    1. Paraformaldehyde (PFA) (Sigma, catalog number: 158127 )
    2. Sucrose (Sigma, catalog number: S8501 )
    3. PBS (Fisher Bioreagents, catalog number: 10649743 )
    4. PFA (see Recipes)

Software

  1. FIJI (https://fiji.sc) or ImageJ (NIH, https://imagej.nih.gov/ij/, version 1.51 was used)
  2. Matlab (MathWorks, version R2017a was used)
  3. SoftWoRks software v6 (or suitable image acquisition software for the microscope to be used)
  4. Excel (or any spreadsheet) to collect data prior to analysis.

Equipment

  1. Dumont forceps #5, 11 cm, straight (Fine Science Tools, catalog number: 11254-20 )
  2. Dumont #5/45 forceps, 11 cm, 45 Degree Angle (Fine Science Tools, catalog number: 11251-35 )
  3. Scissors
  4. Incubator
  5. Laminar flow hood
  6. Dissection microscope
  7. Hemocytometer (Fisher, catalog number: 11704939 )
  8. Autoclave
  9. Tissue culture hood
  10. 4 °C refrigerator
  11. -20 °C freezer
  12. 37 °C water bath
  13. Cell culture incubator set at 37 °C, 5% CO2
  14. Orbital shaker (Fisher, catalog number: 10759145 )
  15. DeltaVision Elite microscope

Note: Alternatively, any inverted high-resolution fluorescent microscope can be used.

Procedure

Note: Perform all tissue culture steps in a sterile environment. Carry out dissections in a laminar flow hood on a dissection microscope. Perform microfluidic device preparation, tissue dissociation and cell culture in a vertical flow hood.

  1. Preparation of coverslips and microfluidic devices
    1. Glass coverslips
      1. Place coverslips in a plastic container containing 1 N NaOH and wash for 1 h on an orbital shaker at approximately 120 rpm, careful to avoid spills.
      2. Remove NaOH from the container and wash coverslips with ddH2O several times until the pH returns to neutral.
      3. Store coverslips in 70% ethanol.
    2. Microfluidic devices
      1. Cover the structured surfaces of the device with 3M tape to remove any particulates and fibres.
      2. Peel off the tape and store devices in a container containing 70% ethanol.
      Note: Devices can be stored dry covered with 3M tape or in 70% ethanol for extended periods of time (several months to years).
    3. Assemble devices (1 day before dissection)
      1. Remove two glass coverslips from the 70% ethanol-containing container and place into a sterile Petri dish using forceps.
      2. Allow the glass to dry (~5 min in open air in hood) before applying 1 ml of 0.1 mg/ml PDL onto each the glass for at least 1 h to overnight (Figure 1).


        Figure 1. Coating of glass coverslips

      3. Remove PDL solution and wash the glass with autoclaved ddH2O three times.
      4. Allow the PDL-coated glass to air-dry inside a sterile tissue culture hood for ~1 h prior to the addition of the microfluidic device.
        Note: Glass coverslips that have been PDL-coated, washed and dried can be stored in Petri dishes at -20 °C for up to two weeks.
      5. Remove the devices from the 70% ethanol and place the devices at an angle on the edge of a sterile Petri dish, channel-side up (i.e., upside down, see Figure 2A).


        Figure 2. Assembly of microfluidic devices. (A, B) Schematic of the setup. A. The devices are dried groove-side up in a 10 cm Petri dish in a sterile environment. B. They are then assembled with the structured side facing the glass coverslip, gently pushing the device down in the non-structured areas to allow good contact for leak-free sealing between PDMS and the glass substrate. (C, D) Photos of two assembled devices. C. Dry devices before media addition. D. Two filled devices showing one well sealed (top) and a leaky (bottom) device. Devices were filled with food dye for better contrast.

      6. Allow the devices to completely dry (~1 h).
      7. Using sterile forceps, set down the dry devices (channel-side down) on to the centre of the PDL-coated glass coverslips. Use the blunt end of the forceps to gently press down the devices onto the coverslips to ensure even PDMS-glass contact, removing any air pockets (see Figure 2B).
      8. Observe that the devices adhere to the surface of the glass, ensuring a tight seal.
      9. Add 10 µl of 70% ethanol to inlets at one side of the device and observe as the channels fill from one port to the next.
      10. Place lid on the Petri dish and examine the assembled device on a light microscope making sure that there is no leakage or bubbles trapped inside the channels and/or microgrooves.
      11. Next, rinse out ethanol by adding 50 µl of supplemented Neurobasal medium to each channel, allowing the device to fill. Remove ethanol from outlet and top up inlets with supplemented Neurobasal medium. Place in the incubator overnight.
      Note: Perform these steps in quick succession to ensure liquid in device does not evaporate.

  2. Hippocampal dissection and primary cell preparation
    1. Sacrifice the pregnant female using CO2 followed by cervical dislocation. Remove the uterus, place it into sterile Petri dish filled with PBS and move to a horizontal flow hood for fine dissection
    2. Isolate the embryos and collect into fresh PBS. Make sure to rinse off remaining blood.
    3. Isolate embryonic brains. Using a dissection microscope, remove the meninges and dissect out the hippocampi. On average, one embryo yields ~500,000 hippocampal neurons, with between 4-10 embryos per pregnant female.
      Note: A helpful illustration of murine embryonic hippocampal dissection can be found at Seibenhener and Wooten, 2012.
    4. Collect the isolated hippocampi in 1 ml PBS into a 15 ml conical tube and move to a vertical flow hood.
    5. Add 100 µl Trypsin-EDTA and incubate in a water bath at 37 °C for 8 min to disrupt cell-cell contacts.
    6. Add 1 ml serum to inactivate the trypsin and spin cells for 3 min at 300 x g. Remove supernatant and resuspend cells in 1 ml culture medium.
    7. Triturate cells with a 1 ml pipette about 6-8 times. This will dissociate tissue into a single cell suspension.
      Note: If the cell suspension begins to clump (often observed with older embryos), add 50 µl DNAse solution for trituration.
    8. Filter the dissociated cells through a 40 µm cell strainer to remove non-dissociated larger chunks and count cells using a hemacytometer.

  3. Cell Culture
    1. Transfer 300,000 cells into an Eppendorf tube.
      Note: If you plate more than 2 channels per device adjust cell number and media volume accordingly. Use 60,000-70,000 cells per channel.
    2. Spin the tube at 300 x g for 3 min. 
    3. Gently resuspend the cell pellet in the Eppendorf tube in 50 µl complete NBM.
    4. Remove the primed devices from the incubator and transfer into a sterile culture hood.
    5. Remove all the media from all of the inlets in the device.
    6. Starting from the left port (Al) add 10 µl of cell suspension and remove 1 µl from the adjacent connecting right port (Ar) to encourage flow of cells (see Figure 3A). 
    7. Close the Petri dish and use a light microscope to check that cells have entered the main culture channels. Return Petri dish into a sterile hood.
    8. Continue on to fill the other channels (e.g., Bl) as above.
    9. Allow the cells to settle close to the microgrooves by propping the Petri dish containing the device at an angle (see Figure 3B). After 5-10 min transfer the Petri dishes to the incubator for an additional 45 min to allow the cells to adhere to the glass.
    10. Top up medium by adding 50 µl of complete NBM to all the ports.
    11.  Add 1 ml of sterile ddH2O on the side of the Petri dish to maintain humidified atmosphere before replacing the lid and place cells in incubator. This counts as day in vitro (DIV) 0.


      Figure 3. Plating of neurons into the device. A. Add neuronal suspension into port, as close to the channel as possible, and encourage flow into the channel by removing a small volume from the other side. B. Once plated, place devices at a gentle angle so that gravity guides the cells to settle near the microchannels. For this, the culture channels should be horizontal with channel A on top, so that cells in culture channel A settle near the microgrooves connecting to channel B and those in channel B near the microgrooves connecting to channel C.

  4. Transfection
    On the following day (DIV1), transfect the cultures in the devices with the relevant constructs. To assay tau propagation, we transfected human tau 0N4R constructs described in Hoover et al., 2010 as follows: GFP-tauE14 in channel A, and RFP-tauWT in channel B (Figure 4A).
    1. Add 25 µl of OptiMEM each in two separate Eppendorf tubes, labeled 1 and 2.
    2. Add 0.5 µg of plasmid in tube 1 and 0.5 µl Lipofectamine2000 in tube 2. Mix by pipetting up and down.
    3. Make the transfection reagent by mixing tube 1 and 2 by gentle bubbling. Do not vortex. Allow to stand inside a sterile hood for 20 min.
    4. Take the device from the incubator into a sterile TC hood and remove all media in both ports of channel A (the channel to be transfected) (see Figure 4B for workflow).
    5. Top up ports connected to culture channel B with complete NBM. A high volume of media within the ports of channel B will fluidically isolate this channel and prevent flow of the transfection reagent from channel A to channel B.


      Figure 4. Transfection within the device. A. Diagram of the transfected device. Individual presynaptic neurons in channel A express mutant tau (green), while the majority of cells remain untransfected (blue). Transfected projecting neurons make contact with postsynaptic neurons expressing fluorescent wildtype tau. B. Workflow of the transfection procedure. C. Partial reconstruction of two transfected, connecting neurons within a device. Scale bar = 10 µm.

    6. Add 50 µl transfection reagent on the left feeding port of channel A.
    7. Close the Petri dish lid and incubate for 40 min in the incubator at 37 °C.
    8. Remove all media from ports A and add 50 µl of fresh complete NBM in all the left ports to flush remove the remaining transfection reagent.
    9. Leave for 2 min before removing excess media in the right port.
    10. Now, add 50 µl of complete NBM to all ports.
    11. Repeat to transfect channel B.
    12. Add 1 ml sterile ddH2O on the side of the Petri dish to prevent drying out before placing back into the incubator overnight.
    13. Feed cultures every 2-3 days by topping up one port. Remove excess media from opposite port as necessary.
    Note: Replenish the 1 ml sterile ddH2O in the Petri dish every 2-3 days to prevent drying out of cultures.

  5. Fixation
    Note: Devices can be imaged live in phenol red-free media, or fixed for later observation. We typically fix the devices unless observing dynamic processes.
    1. On the desired day in vitro, stop the experiment by fixing cells.
    2. Remove the media from all ports.
    3. Fill port Ar with 50 µl PFA. Allow flow into the main channel and through the microgrooves.
    4. Top up port Ar and fill port Br.
    5. Leave to fix for 15 min.
    6. Remove PFA solution and fill device with PBS or TBS.
    7. Fixed devices can be stored short-term in the fridge (up to 2 weeks), but ensure that they do not dry out by topping ports up with PBS or TBS.

  6. Microscopy
    Note: while we performed the imaging on a DeltaVision microscope, any high resolution inverted fluorescence microscope is suitable. We typically use a 60x oil objective, and 1 pixel = 0.1075 µm.
    1. Clean bottom of the glass coverslip with 70% EtOH.
    2. Image cells on an inverted fluorescence microscope using a 60x oil objective and adequate filter settings for the GFP and RFP channels.
    3. For every connecting pair, ensure you image the corresponding cell bodies and distal axonal segments. Ideally capture corresponding differential interfering contrast (DIC) or phase image for quality control.
      Notes:
      1. For the analysis you will need an axonal stretch that is in focus for a length of ≥ 75 µm.
      2. Any axonal stretch that is ≥ 300 µm from the cell body can be used as “distal axon”. Ensure you only use one axonal stretch per neuron for analysis.
      3. Ensure you do not image blebbed cells as this will interfere with the analysis (see Figure 5). In doubt check axonal integrity on transmitted light channel to ensure that the axon structure is smooth.


        Figure 5. Examples of two blebbed axonal stretches expressing GFP-TauE14 that should be excluded from analysis. Scale bar = 10 µm.

    4. Capture image at highest pixel depth possible (we used 16bit).

Data analysis

  1. Reading out fluorescence intensities along the axon in ImageJ/FIJI
    Note: The data analysis is based on the observation that aggregation of fluorescent tau leads to a fluorescence redistribution within the cell, with brighter spots and stretches appearing that represent accumulation (Figures 5 and 9).
    1. Open original microscopy file of a distal axonal segment in FIJI or ImageJ.
    2. To create a plot profile on the opened image, right click on the line tool (Figure 6) and select “Segmented Line” in the drop-down menu (Figure 6A).
    3. Create a segmented line plot along the length of the axon (Figure 6B). Double-click to stop the creating a segmented line.


      Figure 6. Trace the axon using the segmented line tool

    4. To quantify the fluorescence values along the axon that has been plotted, go to Analyze→Plot Profile (Figure 7). This will generate a list of pixel intensity values corresponding to the length of the axon that was plotted (Figure 8).


      Figure 7. Generate a plot profile to read out fluorescence intensity values along the length of the axon


      Figure 8. To save the values for further analysis, click on List and Select All values to copy. Collect as columns in a spreadsheet, ensuring you keep track which column of data belongs to which original axonal image.

    5. Click on the “List” (as shown in the blue arrow in Figure 8) to view the individual values. To save the data: Select all→Copy.
    6. Paste into columns in an excel spreadsheet.
    7. Figure 9 shows an example of an analysed stretch next to its plot profile.


      Figure 9. Example of an imaged axon and its corresponding plot profile

    Notes:
    1. Paste all measured stretches from one experiment into one file. Use one spreadsheet per condition.
    2. You will initially also need one spreadsheet with a range of control values from different time points.

  2. Fluorescence distribution analysis
    Note: To begin this part of the analysis you need a range of “control” axons from different experimental sets across the time points analysed. We recommend a minimum of 30 imaged axonal stretches. This will determine the allowed baseline variability within your experimental data. It also allows for individual control axons to score positive in the later analysis.
    1. Establishing the allowed baseline variation across experiments
      1. Copy a random selection of values from control axons across different experiments into a spreadsheet.
      2. Open the MATLAB script “baseline” found at https://www.mathworks.com/matlabcentral/fileexchange/74503-baseline-analysis.
      3. Import data into the MATLAB script and run the script.
      4. The script will first ensure that different expression levels in individual cells do not skew the analysis by substracting the 10th percentile values from each series (Figure 10).
        Note: Explanation of script “baseline” (https://www.mathworks.com/matlabcentral/fileexchange/74503-baseline-analysis). This script is designed to determine the signal variability within the controls across biological repeats and time points. For this, you need sample control axons randomly picked from the different experiments and paste these into individual columns in an excel spreadsheet. When you run the script it imports the columns as “data”. First, the 10th percentile from each column is determined and deducted to eliminate variability due to different expression levels. The result is designated “zero”. These values are then listed in a single column, and the mean and standard deviation are determined. Choose mean plus 5 standard deviations as allowed baseline variation for script “axon_analysis”.


        Figure 10. Visual representation of the first part of the data analysis, determining allowed baseline variability. In all analysis, different expression levels (as seen in raw plot profiles displayed in A) are taken into account by substracting the 10th percentile of all values in each trace (the result is displayed in B). The cutoff determined in figure 10 results in the grey shaded box in B and allows for control axons (such as the black trace) to score positive in the later analysis.

      5. It will then determine the average fluorescent intensity and the standard deviation. Choose mean plus 5 standard deviations as allowed baseline variation. This ensures your analysis is robust against minor variations in focus along the axonal stretch.

    2. Scoring aggregation within one experiment
      1. Use MATLAB script “axon_analysis” found here.
        Note: Explanation of the script “axon_analysis” (available here). This script is designed to detect increased variability in fluorescence distribution, which is observed upon formation of fluorescent accumulations/ aggregates. You need paired control and experimental axons from one repeat, the values pasted as columns into two separate spreadsheets. Important: adjust the number in line 25 to the number determined following script “baseline_analysis”. The script imports your control (e.g., wildtype tau) values as “control” and your experimental (e.g., mutant tau) values as “experimental”. As above, all values are zeroed to the 10th percentile to eliminate variability due to different expression levels (lines 29, 32 and 64, 67). The first part of the script determines the variability within this control dataset. To allow for control axons to score positive, only those axons falling within the allowed baseline variability determined in the script “baseline_analysis” are included (line 25). A logical array (line 51) then tests for each individual fluorescent value whether it is larger than the mean plus 5 standard deviations of those axons that were included into the control group in line 41. Those that score positive in each column are assigned a “1” and are added up and divided by the total number of individual values per column. This gives a fraction of axonal values scoring positive. If this fraction is > 0.1 (10% of the values) the axon is counted as positive. Line 60 determines the percentage of control axons meeting this criteria.
        The second part of the script tests the experimental dataset. Following deduction of the 10th percentile (lines 64, 67), the script tests all individual values against the maximum allowed baseline value determined from the controls (line 71), adds up the positive scores from each axon (line 74), normalizes these to the size of the dataset (lines 77, 78) to determine the percentage of axons scoring positive in this experiment (line 80).

      2. Adjust in line 25 to the value determined as allowed baseline variation above.
      3. Import control and experimental data set from one experiment as “control” and “experimental”.
      4. The script will first determine the observed variation of all axons that are within the allowed variability across experiments in this particular experiment (Figure 10B, grey shaded area), and then use this value to determine the percentage of control axons that have more than 10% of their values outside the allowed variability and thus score positive (such as the black trace in Figure 10B).
      5. It will then determine the percentage of experimental axons that have more than 10% of their values outside the allowed variability and thus score positive (see Figure 11).


        Figure 11. Visual representation of the aggregate analysis. First, intensity values outside the allowed variability are determined (A, values outside the grey box). Then the script calculates what percentage of axonal stretch falls within this category. Results for individual axons are plotted in B. The axons are scored as positive if more than 10% of their values are in this category. This allows to rule out false-positives such as due to untransfected axons crossing. Finally, the percentage of axons that score positive within one experiment is calculated.

      6. You now have the results for one repeat.

Notes

  1. Transfection efficiency is generally low, expect ~2-3 transfected neurons that also project through the microgrooves to the adjacent channel per device.
  2. Each axon that projects through to the next channel typically branches out widely, thus almost every projecting neuron will make contact with a transfected neuron in the next channel.
  3. The low transfection efficiency allows for full reconstruction of the two connecting neurons. Viral transduction may be used, but its generally high efficiency hampers the ability to identify individually connecting neurons and thus is more useful for biochemical approaches.
  4. In our hands the aggregation analysis was highly reproducible between different preparations, as well between individuals performing the experiment.
  5. We typically observed aggregation, measured as formation of dense fluorescent puncta, from DIV8 onwards.
  6. Neurons containing fluorescent aggregates are positive for misfolded tau (MC1 staining), but due to low transfection efficiency we have not performed biochemical analysis to better define the nature of these aggregates.

Recipes

  1. PDL solution
    0.1 mg/ml poly-D-lysine in sterile H2O
    Can be stored frozen as 10 mg/ml stock or 0.1 mg/ml working aliquots
  2. Culture medium (complete NBM)
    Fully supplemented Neurobasal medium
    To 49 ml Neurobasal medium, add 1 ml B27 and 125 µl glutamax or glutamine
  3. DNase solution
    Resuspend lyophilised powder to 1 mg/ml in neurobasal medium and store frozen in aliquots
  4. PFA
    4% paraformaldehyde/20% sucrose in PBS

Acknowledgments

This work was supported by Alzheimer's Research UK (Grants ARUK-PhD2014-10 and ARUK-PPG2017B-001), the Gerald Kerkut Charitable Trust and the Biotechnology and Biological Sciences Research Council (BB/L007576/1). This is the detailed protocol underlying the propagation work published in Hallinan et al., 2019b.

Competing interests

The authors declare no competing financial interests.

Ethics

All experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986 set out by the UK Home Office.

References

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  13. Takeda, S., Wegmann, S., Cho, H., DeVos, S. L., Commins, C., Roe, A. D., Nicholls, S. B., Carlson, G. A., Pitstick, R., Nobuhara, C. K., Costantino, I., Frosch, M. P., Muller, D. J., Irimia, D. and Hyman, B. T. (2015). Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer's disease brain. Nat Commun 6: 8490. 
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  15. Wu, J. W., Herman, M., Liu, L., Simoes, S., Acker, C. M., Figueroa, H., Steinberg, J. I., Margittai, M., Kayed, R., Zurzolo, C., Di Paolo, G. and Duff, K. E. (2013). Small misfolded Tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons. J Biol Chem 288(3): 1856-1870.
  16. Wu, J. W., Hussaini, S. A., Bastille, I. M., Rodriguez, G. A., Mrejeru, A., Rilett, K., Sanders, D. W., Cook, C., Fu, H., Boonen, R. A., Herman, M., Nahmani, E., Emrani, S., Figueroa, Y. H., Diamond, M. I., Clelland, C. L., Wray, S. and Duff, K. E. (2016). Neuronal activity enhances tau propagation and tau pathology in vivo. Nat Neurosci 19(8): 1085-1092.

简介

[摘要] 错折叠的聚集性tau蛋白的沉积是几种神经退行性疾病的标志,统称为“ tauopathies”。Tau病理以a病毒样方式沿着连接的路径在整个大脑中传播。tau病理学在整个电路中传播的过程是一项深入研究的重点,并且已在人体死后大脑和疾病小鼠模型中进行了体内研究,在包括原代神经元在内的各种细胞系统中进行了体外研究,并使用纯化的重组tau蛋白。在这里,我们描述了一种协议,该协议利用微流控设备中排列的简约神经元回路来跟踪tau错折叠从突触前神经元向突触后神经元的传播。该方法允许高分辨率成像以及释放和接收神经元的单独操作,因此有利于研究tau和其他错折叠蛋白的体外繁殖。

[背景 ] 错折叠的蛋白质在整个大脑中的传播是病理学在多种神经退行性疾病中传播的基础,包括阿尔茨海默氏病,帕金森氏症和Pri病毒病(Goedert 等人,2010;Davis 等人,2018; Hallinan 等人,2019a )。了解潜在机制可能有助于限制疾病进展,因此是深入研究的领域。存在几种体内和体外测定法来监测该过程,包括对过度表达的突变型人tau产生神经变性的小鼠模型(Allen 等人,2002; Ramsden 等人,2005; Santa Cruz 等人,2005; Gibbons 等人。等人,2017)。体外模型包括表达可监测聚集的生物传感器的细胞系(Kfoury 等,2012; Holmes 等,2014)和原代神经元培养,包括微流控装置中的培养(Wu 等,2013 和2016,Takeda 等。,2015)。在此协议中,我们将详细描述体外分析,以监测tau错折叠跨连接神经元的播种和繁殖。该协议建立在如上所述的先前建立的测定之上,但是还不仅可以监视错误折叠的tau从一个神经元传到另一个神经元的过程,而且还可以监视先前未受影响的细胞中的接种情况,从而完全概括神经元间的繁殖。微流体设置进一步允许对突触前和突触后细胞进行单独操作,并对两个连接的神经元进行高分辨率的活细胞和固定细胞成像。该测定法可以适合于研究其他蛋白病的传播,例如病毒或α-突触核蛋白的病理学。此外,基本的定向和连接设置允许培养一系列不同的神经元亚型,从而研究各种其他与运输和传播有关的神经毒性剂,包括病毒和环境侮辱性神经毒性剂,例如纳米颗粒毒性。

关键字:蛋白质错误折叠传播, 微流体装置, 海马体培养, Tau, 成像, 图像分析

材料和试剂


 


A. 微流体装置的准备      


1. 微流体装置      


注意:微流体装置可从Xona Microfluidics商购获得,或者可以按照Taylor等人在2005年和Holloway等人在2019年所述的方法制造。对于单向增长,我们建议使用Holloway 等人所述的“箭头”设备。,2019 。


2. 22 x 50毫米矩形盖玻片(厚度为0.16-0.19毫米,Smith Scientific,目录号:NPS16 / 2250)       


3. 94/16毫米P etri盘,无通气(Greiner Bio-one,目录号:632180)       


4. 3 M透明胶带       


5. 聚-D- 赖氨酸氢溴酸盐(PDL,西格玛,目录号:P7886)       


6. 无菌ddH 2 O       


7. 70%乙醇(EtOH)       


8. 神经基础培养基(Gibco,目录号:21103-049)       


 


B. 原代神经元培养和转染      


15 ml猎鹰锥形管17 / 120MM (Cellstar,目录号188271)
50毫升猎鹰锥形管115分之30(蜂星,目录号:227261)
40 sterm无菌细胞过滤器(Fisher Scientific,目录号:15360801)
定时怀孕雌性小鼠E15-E18
注:w ^ ê经常使用C57BL / 6小鼠。


Glutamax 补充剂(Gibco,目录号:35050-038)
神经基底介质(NBM)(Gibco,目录号:21103-049)
胎牛血清(FBS)(Gibco,目录号:10270-106)
Dulbecco的改良版Eagle Medium(DMEM)(Gibco,目录号:41965-039)
的Opti-MEM还原血清培养基glutamax的补充物(Gibco公司,目录号:51985-026)
DNase(Sigma,目录号:DN25;可选)
B27 Media Supplement,50x(Gibco,货号:17504-044)
Lipofectamine2000(Gibco,目录号:1168027)
0.5%胰蛋白酶-EDTA(Gibco,目录号:15400-054)
磷酸盐缓冲盐溶液(DPBS),无钙(Ca 2+ )和镁(Mg 2 + )(Gibco,目录号:14190-094)
PDL 解决方案(请参阅食谱)
培养基(完全NBM)(请参阅食谱)
DNase 解决方案(请参阅食谱)
 


C. 固定和成像      


多聚甲醛(PFA)(西格玛,目录号:158127)
蔗糖(Sigma,目录号:S8501)
PBS(Fisher Bioreagents,目录号:10649743)
PFA(请参阅食谱)
 


软件


 


FIJI(https://fiji.sc)或ImageJ(使用NIH,https: //imagej.nih.gov/ij/ ,版本1.51 )
Matlab(使用MathWorks 版本R2017a )
SoftWoRks 软件v6(或要使用的显微镜的合适图像采集软件)
Excel(或任何电子表格)在分析之前收集数据。
 


设备


 


1. Dumont镊子#5,11厘米,直的(Fine Science Tools,目录号:11254-20)       


2. Dumont#5/45镊子,11厘米,45度角(精细科学工具,目录号:11251-35)       


3. 剪刀       


4. 孵化器       


5. 层流罩       


6. 解剖显微镜       


7. 血细胞计数器(Fisher,目录号:11704939)       


8. 高压釜       


9. 组织培养罩       


10. 4°C冰箱   


11.- 20°C冰柜   


12. 37°C水浴   


13. 细胞培养箱设置为37°C,5%CO 2   


14. 轨道振动筛(Fisher,目录号:10759145)   


15. DeltaVision Elite显微镜   


注意:或者,可以使用任何倒置的高分辨率荧光显微镜。


 


程序


 


注意:在无菌环境中执行所有组织培养步骤。在解剖显微镜的层流罩中进行解剖。在垂直流罩中进行微流控设备准备,组织解离和细胞培养。


A. 盖玻片和微流体装置的制备      


1. 玻璃盖玻片       


将盖玻片放入装有1 N NaOH的塑料容器中,并在定轨摇床上以大约120 rpm的速度洗涤1小时,注意避免溢出。
从容器中取出NaOH,并用ddH 2 O 清洗盖玻片数次,直到pH恢复到中性。
将盖玻片储存在70%的乙醇中。
2. 微流体装置      


用3M胶带覆盖设备的结构化表面,以除去任何微粒和纤维。
剥下胶带,将设备存储在包含70%乙醇的容器中。
注:d evices可以存储干覆盖有3M胶带或在70%乙醇中的时间过长(数月至数年)。


3. 组装器械(解剖前1天)      


从含70%乙醇的容器中取出两个玻璃盖玻片,并用镊子将其置于无菌培养皿中。
让玻璃干燥(在通风橱中约5分钟),然后将1 ml的0.1 mg / ml PDL涂在每个玻璃上至少1 h至过夜(图1)。
 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图1.jpg


图1.玻璃盖玻片的涂层


 


除去PDL溶液,并用高压灭菌的ddH 2 O 次洗涤玻璃。
在添加微流控设备之前,让涂有PDL的玻璃在无菌组织培养罩内风干〜1 h。
注:g ^ 已PDL涂覆,洗涤和干燥小姑娘盖玻片可以在-20存储在培养皿℃下进行长达两周。


从70%的乙醇中取出设备,将设备倾斜放置在无菌培养皿的边缘,通道朝上(即,倒置,见图2A)。
 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图2.jpg


图2. 微流控设备的组装。(A,B)设置示意图。答:将设备在无菌环境中的10厘米培养皿中槽侧朝上干燥。B.然后将其组装,使结构化的一面朝向玻璃盖玻片,将设备轻轻向下推入非结构化区域,以实现良好的接触,从而实现PDMS与玻璃基板之间的无泄漏密封。(C,D)两个组装好的设备的照片。C.在添加介质之前干燥设备。D.两个填充的设备,其中一个密封良好(顶部),一个泄漏(底部)设备。设备中填充了食用染料,以实现更好的对比度。


 


让设备完全干燥(〜1小时)。
使用无菌镊子,将干燥装置(通道朝下)放在PDL涂层的玻璃盖玻片的中央。用镊子的钝端轻轻将设备向下按到盖玻片上,以确保PDMS-玻璃均匀接触,并去除任何气穴(请参见图2B)。
观察设备是否粘附在玻璃表面上,确保紧密密封。
将10分之70%的乙醇添加到设备一侧的入口,观察通道从一个端口到另一个端口的填充情况。
将盖子放在培养皿上,并在光学显微镜上检查组装的设备,确保通道和/ 或微槽内没有泄漏或气泡。
接下来,通过向每个通道中添加50ul补充的Neurobasal培养基冲洗出乙醇,以填充设备。从出口除去乙醇,并用补充的Neurobasal培养基补足进口。放在培养箱中过夜。
注:P erform临门这些步骤,以确保设备不会蒸发的液体。






B. 海马解剖和原代细胞的制备      


1. 用CO 2 牺牲孕妇,然后进行颈脱位。取出子宫,放入装有PBS的无菌培养皿中,移至水平流罩进行精细解剖      


2. 分离胚胎并收集到新鲜的PBS中。确保冲洗掉剩余的血液。      


3. 隔离胚胎大脑。使用解剖显微镜,去除脑膜并解剖出海马。平均而言,一个胚胎产生约500,000个海马神经元,每个怀孕的雌性有4-10个胚胎。      


注:一个小鼠胚胎海马解剖有用的例证,可以发现Seibenhener和伍滕,2012 。


4. 将分离的海马以1 ml PBS收集到15 ml锥形管中,并移至垂直流罩。      


5. 加入100胰胰蛋白酶-EDTA,并在37的水浴中孵育       ℃ 8分钟以破坏细胞间的接触。


6. 加入1 ml血清使胰蛋白酶失活,并以300 xg的转速旋转细胞3分钟。除去上清液并将细胞重悬于1 ml培养基中。      


7. 用1 ml移液器研磨细胞约6-8次。这会将组织分解成单个细胞悬液。      


注意:如果细胞悬液开始结块(通常在年长的胚胎中观察到),请添加50 µl DNAse 溶液进行研磨。


8. 过滤器的解离的细胞通过40μm细胞过滤器以除去未离解的更大的块,并使用计数细胞血细胞计数器。       


 


C. 细胞培养      


1. 将300,000个细胞转移到Eppendorf管中。      


注意:如果您为每个设备延迟两个以上的频道,请相应地调整单元号和媒体音量。每个通道使用60,000-70,000个单元。


2. 以300 xg 旋转试管3分钟。       


3. 轻轻地将细胞沉淀重悬在Eppendorf管中的50个完整NBM中。       


4. 从培养箱中取出准备好的设备,并转移到无菌培养罩中。       


5. 从设备的所有入口中取出所有介质。       


6. 从左侧端口(A l )开始,添加10µl细胞悬液,并从相邻的右侧连接端口(A r )中移除1个,以促进细胞流动(请参见图3A)。       


7. 关闭培养皿,并使用光学显微镜检查细胞是否已进入主要培养通道。将培养皿倒入无菌罩中。       


8. 继续上以填充其它信道(例如,B 升)如上述。       


9. 通过以一定角度支撑装有该设备的培养皿,使细胞靠近微槽沉降(请参见图3B)。5-10分钟后,将培养皿再移至培养箱中45分钟,以使细胞粘附在玻璃板上。      


10. 通过向所有端口中添加50份完整的NBM来补充培养基。   


11. 在盖上培养皿之前,在培养皿侧面上加入1 ml无菌ddH 2 O,以保持湿润的气氛,然后盖上盖子并将细胞放在培养箱中。这算作体外天数(DIV)0。   


 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图3.jpg


图3.将神经元电镀到设备中。A.将神经元悬液添加到端口中,并尽可能靠近通道,并通过从另一侧移走少量体积来鼓励流入通道。B.接种后,将设备平缓放置,以使重力引导细胞沉降到微通道附近。为此,培养通道应水平放置,通道A在顶部,以便培养通道A中的细胞沉降在连接至通道B的微槽附近,而通道B中的细胞沉降在连接至通道C的微槽附近。


 


D. 转染      


在第二天(DIV1),用相关构建体转染装置中的培养物。为了测定tau的繁殖,我们转染了Hoover 等人所述的人tau 0N4R构建体。,2010年如下:通道A中的GFP-tau E14 ,通道B中的RFP- tau WT (图4A)。


1. 在两个分别标记为1和2的Eppendorf管中分别加入25的OptiMEM 。       


2. 在试管1中加入0.5 µg质粒,在试管2中加入0.5 Lil Lipofectamine2000。       


3. 轻轻鼓泡,将混合管1和2制成转染试剂。不要涡旋。允许在无菌通风橱中放置20分钟。       


4. 将设备从培养箱中带入无菌TC罩中,并除去通道A(要转染的通道)两个端口中的所有培养基(工作流程请参见图4B)。       


5. 用完整的NBM加满连接到培养通道B的端口。通道B的端口内的大量介质将使该通道流体隔离,并阻止转染试剂从通道A到通道B的流动。       


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图4.jpg


图4.设备内的转染。A.转染设备的示意图。通道A中的单个突触前神经元表达突变体tau(绿色),而大多数细胞仍未转染(蓝色)。转染的投射神经元与表达荧光野生型tau的突触后神经元接触。B.转染程序的工作流程。C.设备中两个转染的连接神经元的部分重建。比例尺= 10 µm。


 


6. 在通道A 的左进料口中添加50 transl转染试剂。       


7. 关闭培养皿盖,并在37°C的培养箱中孵育40分钟。       


8. 从端口A取出所有培养基,并在所有左侧端口中加入50 毫升新鲜的完整NBM以冲洗除去剩余的转染试剂。       


9. 放置2分钟,然后再从右侧端口中取出多余的介质。       


10. 现在,将50个完整的NBM添加到所有端口。   


11. 重复以转染通道B。   


12. 在培养皿侧面上加入1 ml无菌ddH 2 O,以防止变干,然后再放回培养箱中过夜。   


13. 补满一个端口,每2-3天喂一次培养物。如有必要,请从对面的端口中取出多余的介质。   


注意:每2-3天在P etri皿中补充1 ml无菌ddH 2 O,以防止培养物变干。


E. 固定      


注意:设备可以在不含酚红的介质中实时成像,也可以固定以备后用。我们通常会修复设备,除非观察动态过程。


1. 在体外所需的一天,通过固定细胞停止实验。      


2. 从所有端口中取出介质。      


3. 用50 Pl PFA 填充端口A r 。允许流入主通道并通过微槽。      


4. 充值端口一个[R 及填充口B [R 。      


5. 静置15分钟。      


6. 取出PFA溶液,并用PBS或TBS填充设备。      


7. 固定设备可以在冰箱中短期存放(最多2周),但请通过用PBS或TBS填充端口,以确保它们不会变干。      


 


F. 显微镜      


注意:虽然我们在DeltaVision 显微镜上进行成像,但任何高分辨率倒置荧光显微镜都是适用的。我们通常使用60倍油镜,而1个像素= 0.1075 µm。


1. 用70%的乙醇清洁玻璃盖玻片的底部。      


2. 在倒置荧光显微镜上使用60倍油镜和适当的GFP和RFP通道滤镜设置的成像单元。      


3. 对于每个连接对,请确保对相应的细胞体和远端轴突节段成像。理想情况下,捕获相应的差分干扰对比度(DIC)或相位图像以进行质量控制。      


笔记:


为了进行分析,您需要轴突伸展的长度≥75 µm。
距细胞体≥300 µm的任何轴突伸展都可以用作“远端轴突”。确保每个神经元仅使用一个轴突伸展进行分析。
确保您不对气泡细胞成像,因为这会干扰分析(请参见图5)。不确定地检查透射光通道上的轴突完整性,以确保轴突结构光滑。
 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图5.jpg


图5. 表达GFP-Tau E14 的两个起泡的轴突拉伸的例子,应从分析中排除。比例尺= 10 µm。


 


4. 以尽可能高的像素深度捕获图像(我们使用了16位)。       


 


数据分析


 


在ImageJ / FIJI中读取沿轴突的荧光强度
注意:数据分析是基于以下观察结果:荧光tau的聚集会导致细胞内的荧光重新分布,出现亮点和拉伸段代表积累(图5和9)。


打开FIJI或ImageJ中远端轴突节段的原始显微镜文件。
要在打开的图像上创建绘图轮廓,请右键单击线条工具(图6),然后在下拉菜单中选择“分段线”(图6A)。
沿着轴突的长度创建分段线图(图6B)。双击以停止创建分段线。
 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图6.jpg


图6.使用分段线工具跟踪轴突


 


要量化已绘制的轴突周围的荧光值,请转到AnalyzePlot Profile(图7)。这将生成与绘制的轴突长度相对应的像素强度值列表(图8)。
 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图7.jpg


图7.生成图谱以读取沿轴突长度的荧光强度值


 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图8.jpg


图8.要保存值以进行进一步分析,请单击List并选择All values以进行复制。在电子表格中收集为列,以确保您跟踪哪个数据列属于哪个原始轴突图像。


 


单击“列表”(如图8中的蓝色箭头所示)以查看各个值。保存数据:全选 复制。
粘贴到Excel电子表格的列中。
图9显示了其图轮廓旁边的分析拉伸示例。
 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图9.jpg


图9.轴突成像示例及其相应的绘图轮廓


 


笔记:


将来自一个实验的所有测得的拉伸值粘贴到一个文件中。每个条件使用一个电子表格。
您最初还需要一个电子表格,其中包含来自不同时间点的一系列控制值。
 


荧光分布分析
注意:首先,你需要一系列的来自全国各地点的时间不同实验组“控制”轴突的这部分分析分析。我们建议至少30个成像的轴突拉伸。这将确定实验数据中允许的基线变异性。它还允许单个控制轴突在以后的分析中得分为阳性。


在整个实验中建立允许的基线变化
将跨不同实验的控制轴突中随机选择的值复制到电子表格中。
打开https://www.mathworks.com/matlabcentral/fileexchange/74503-baseline-analysis上的MATLAB脚本“基准” 。
将数据导入MATLAB脚本并运行该脚本。
该脚本将首先通过从每个系列中减去第10 个百分位数来确保单个细胞中不同的表达水平不会使分析产生偏差(图10)。
注意:脚本“基准”的说明(https://www.mathworks.com/matlabcentral/fileexchange/74503-baseline-analysis)。该脚本旨在确定跨生物学重复和时间点的控件内信号的可变性。为此,您需要从不同实验中随机选择的示例控制轴突,然后将其粘贴到excel电子表格的各个列中。运行脚本时,它会将列导入为“数据”。首先,确定并扣除每列的第10 个百分位数,以消除由于不同表达水平而引起的变异性。结果指定为“零”。然后将这些值列在单个列中,并确定平均值和标准偏差。选择平均值加5个标准差作为脚本“ axon_analysis ”的允许基线变化。


 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图10.jpg


图10.数据分析第一部分的可视化表示,确定允许的基线变异性。在所有分析中,通过减去每条迹线中所有值的第10 个百分位数,来考虑到不同的表达水平(如在A中显示的原始图配置文件中所示)(结果显示在B中)。图10中确定的截止值导致B中的灰色阴影框,并允许控制轴突(例如黑色迹线)在以后的分析中得分为阳性。


 


然后将确定平均荧光强度和标准偏差。选择平均值加5个标准差作为允许的基线变化。这样可以确保您的分析在轴突伸展时对焦点的微小变化方面都具有强大的抵抗力。
 


一项实验中的评分汇总
使用此处提供的 MATLAB脚本“ axon_analysis ” 。
注意:脚本“ axon_analysis ”的说明(在此处可用)。此脚本旨在检测荧光分布中增加的变异性,这在形成荧光累积物/聚集体时可以观察到。您需要一次重复地将对照轴突和实验轴突配对,将值作为列粘贴到两个单独的电子表格中。重要提示:将第25行中的数字调整为根据脚本“ baseline_analysis ” 确定的数字。该脚本将您的对照(例如野生型tau)值导入为``对照'',并将您的实验(例如突变型tau)值导入为``实验性''。如上所述,所有值都被清零到第10个百分位数,以消除由于不同的表达水平而引起的可变性(第29、32和64、67行)。脚本的第一部分确定了此控件数据集中的可变性。为了使控制轴突得分为正,仅包括那些在脚本“ baseline_analysis ”中确定的允许基线变异范围内的轴突(第25行)。然后,逻辑阵列(第51行)会针对每个单独的荧光值测试其是否大于第41行的对照组中包含的轴突的平均值加5个标准差。在每列中得分为正的那些轴突均被指定为“ 1”,然后相加并除以每列中各个值的总数。这使轴突值的分数为正。如果该分数> 0.1(值的10%),则将轴突计为阳性。第60行确定满足此标准的控制轴突的百分比。


脚本的第二部分测试实验数据集。减去第10个百分位(第64、67行)后,脚本将对照控件确定的最大允许基线值测试所有单个值(第71行),将每个轴突的正值加起来(第74行),将其标准化为数据集的大小(第77、78 行),以确定该实验中得分为阳性的轴突的百分比(第80行)。


 


在第25行中将其调整为确定为上述允许的基线变化的值。
从一个实验中导入对照和实验数据集作为“对照”和“实验性”。
该脚本将首先确定在此特定实验中跨实验在所有允许的变异范围内观察到的所有轴突的变化(图10B,灰色阴影区域),然后使用该值确定具有超过10%的对照轴突的百分比它们的值超出允许的变异性,因此得分为正(例如图10B中的黑色轨迹)。
然后它将确定在允许的变异范围之外具有超过其值10%的实验轴突的百分比,从而获得阳性结果(见图11)。
 


D:\重新格式化\ 2020-7-1 \ 2003078--1494 Katrin Deinhardt 837707 \图jpg \图11.jpg


图11.聚集分析的可视化表示。首先,确定允许的变异性之外的强度值(A,灰色框之外的值)。然后,脚本计算出该类别中轴突伸展的百分比。单个轴突的结果绘制在B中。如果超过10%的轴突属于此类别,则将其评为阳性。这样可以排除假阳性,例如由于未转染的轴突交叉。最后,计算在一个实验中得分为阳性的轴突的百分比。


 


现在,您可以得到重复一次的结果。
 


笔记


 


转染效率通常较低,预计〜2-3个转染的神经元也会通过微槽投射到每个设备的相邻通道。
投射到下一个通道的每个轴突通常会广泛分支,因此几乎每个投射的神经元都会与下一个通道中的转染神经元接触。
低的转染效率导致两个连接神经元的完全重建。可以使用病毒转导,但是其通常的高效率阻碍了识别单独连接的神经元的能力,因此对于生化方法更有用。
在我们手中,聚集分析在不同制剂之间以及进行实验的个体之间是高度可重复的。
我们通常观察到从DIV8开始的聚集,以密集的荧光点形成。
含有荧光聚集体的神经元对tau折叠错误(MC1染色)呈阳性,但由于转染效率低,我们尚未进行生化分析以更好地定义这些聚集体的性质。
 


菜谱


 


PDL 解决方案
无菌H 2 O中的0.1 mg / ml聚D-赖氨酸


可以以10 mg / ml的储备液或0.1 mg / ml的工作等分试样冷冻保存


培养基(完全NBM)
完全补充的神经基础培养基


向49 ml神经基础培养基中,加入1 ml B27 和125 gll 谷氨酰胺或谷氨酰胺


DNase 溶液
将冻干粉重悬于神经基础介质中至1 mg / ml,并冷冻保存


PFA
PBS中含4%多聚甲醛/ 20%蔗糖


 


致谢


 


这项工作得到了英国阿尔茨海默氏症研究中心(格兰茨ARUK-PhD2014-10和ARUK-PPG2017B-001),杰拉尔德· 柯尔库特慈善基金会和生物技术与生物科学研究委员会的支持(BB / L007576 / 1)。这是Hallinan 等人发表的有关传播工作的详细协议。,2019b 。


 


利益争夺


 


作者宣称没有任何竞争性的经济利益。


 


伦理


 


所有实验均根据英国内政部制定的1986年动物(科学程序)法进行。


 


参考文献


 


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Copyright: © 2020 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. Hallinan, G. I., Lopez, D. M., Vargas-Caballero, M., West, J. and Deinhardt, K. (2020). Co-culture of Murine Neurons Using a Microfluidic Device for The Study of Tau Misfolding Propagation. Bio-protocol 10(16): e3718. DOI: 10.21769/BioProtoc.3718.
  2. Hallinan, G. I., Vargas-Caballero, M., West, J. and Deinhardt, K. (2019b). Tau misfolding efficiently propagates between individual intact hippocampal neurons. J Neurosci 39(48): 9623-9632.
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