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Explant Methodology for Analyzing Neuroblast Migration
采用外植体方法分析神经母细胞迁移   

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Abstract

The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration.

Keywords: Neuroblasts(神经母细胞), Neural stem cells(神经干细胞), SVZ-derived explants(SVZ源性外植体), Rostral migratory stream(嘴侧迁移流), Matrigel(基质胶), in vitro(体外)

Background

The adult mammalian forebrain contains a neurogenic niche that lies alongside the lateral ventricle in rodents and humans alike, and is aptly named the subventricular zone (SVZ). In rodents, the SVZ is a thin ‘wedge’ of cells, covering the entire wall of the lateral ventricle (Mirzadeh et al., 2010; Paez-Gonzalez et al., 2014; Dixon et al., 2016). Within the SVZ the slow-dividing astrocyte-like type B cells differentiate into rapidly dividing type C neural progenitor cells (also known as transit amplifying cells) that give rise to doublecortin-positive type A neuroblasts, although oligodendrocytes and astrocytes are also capable of being produced (Garcia-Verdugo et al., 1998; Tavazoie et al., 2008; Rikani et al., 2013). In rodents, there are an estimated 10,000 to 30,000 neuroblasts produced daily. These neuroblasts form chains as they migrate through the rostral migratory stream (RMS) to the olfactory bulb (Lois and Alvarez-Buylla, 1994; Sun et al., 2010). Ablation studies suggest it takes approximately 2 days for fast dividing type C neural progenitor cells to populate the SVZ, and an additional 2.5 days for neuroblasts to appear (Doetsch et al., 1999). A small percentage of these neuroblasts are capable of migrating ectopically out of the RMS into surrounding tissues in naïve mice; however, this phenomenon is drastically increased following brain injury (Dixon et al., 2016). The ability of neuroblasts to redirect their migratory routes towards damaged tissues has been shown to have beneficial effects on brain recovery (Li et al., 2010; Dixon et al., 2015), which can occur as early as 3 days post-injury (Ramaswamy et al., 2005; Dixon et al., 2016).

The self-renewal capacity of stem cells in culture was first identified in 1992 by Reynolds and Weiss (Reynolds and Weiss, 1992). The authors used fine dissection to harvest a small piece of the adult mouse striatum, before trypsinizing, dissociating and culturing. This original protocol, and subsequent variations, are now widely used to grow neurospheres or monolayer cultures to assess factors regulating stem cell survival, proliferation and/or differentiation into neurons (Theus et al., 2012). These culturing systems rely on the presence of growth factors (i.e., fibroblast and epidermal growth factors) to maintain proliferative states, whereas the withdrawal of these factors induces rapid differentiation into mature neurons. Unfortunately, these conditions limit the ability to analyze factors that regulate type A neuroblasts, a transient stage between the stem cell and neuron. To counteract this limitation; pieces of SVZ-derived tissue can be harvested and cultured as explants in a Matrigel containing laminin and collagen, which maintains the neural stem cells in their neuroblast state, allowing them to migrate (Ward and Rao, 2005; Dixon et al., 2016). Furthermore, neuroblast migration from cultured SVZ explants has similar characteristics to those observed in the RMS. Here, we describe an RMS explant methodology, modified from Ward and colleague (Leong et al., 2011), used to study chain migration of SVZ-derived neuroblasts.

Materials and Reagents

  1. 35 mm cell culture dishes with 4 internal wells, each with a diameter of 10 mm (Greiner Bio One International, catalog number: 627170 )
  2. 10 cm cell culture dishes (Corning, catalog number: 353003 )
  3. Pre-chilled (-20 °C) tissue culture pipette filter tips
  4. 10 µl tips (Corning, Axygen®, catalog number: TF-300-R-S )
  5. 200 µl tips (Corning, Axygen®, catalog number: TF-200-R-S )
  6. 1,000 µl tips (Corning, Axygen®, catalog number: TF-1000-R-S )
  7. 5 ml serological pipettes and pipette-boy (VWR, catalog number: 612-3702 )
  8. 1.5 ml Eppendorf microcentrifuge tubes (VWR, catalog number: 211-0007 )
  9. 3.2 ml disposable transfer pipette (Thermo Fisher Scientific, catalog number: BER202-1S )
  10. 1 ml insulin syringe with detachable needle (BD, catalog number: 329651 )
  11. Ice tray and ice as available
  12. Small biohazard bags as available
  13. Marker pen (e.g., Sharpie) for labelling cell culture plates
  14. 1-2 postnatal day old C57Bl/6 wildtype mice (Animal Resources Centre, Australia) or from local animal supplier
  15. Absolute ethanol (Sigma-Aldrich, catalog number: 24102 )
  16. Hanks balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14170112 )
  17. Growth factor reduced Matrigel (Corning, catalog number: 356230 )
  18. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
  19. B27 supplement x50 (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  20. 200 mM glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  21. Penicillin/streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  22. Recombinant murine fibroblast growth factor (FGF) basic 1 mg/ml (PeproTech, catalog number: 450-33 )
  23. Recombinant murine epidermal growth factor (EGF) 1 mg/ml (PeproTech, catalog number: 315-09 )
  24. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  25. Sodium phosphate dibasic (Na2HPO4) (Chem Supply, catalog number: SA026 )
  26. Sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) (Chem Supply, catalog number: SO03310500 )
  27. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  28. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
  29. DAPI (Thermo Fisher Scientific, GibcoTM, catalog number: D1306 )
  30. Donkey serum (Sigma Aldrich, catalog number: D9663 )
  31. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  32. Goat anti-doublecortin (DCX) antibody (Santa Cruz Biotechnology, catalog number: sc-8066 or sc-271390 )
    Note: The authors used sc-8066 , this antibody has been discontinued. A suggested alternative from Santa Cruz Biotechnology is sc-271390 .
  33. Cy3-conjugated donkey anti-goat antibody (Jackson ImmunoResearch, catalog number: 705-165-147 )
  34. 80% ethanol solution (see Recipes)
  35. Complete neurobasal medium (see Recipes)
  36. Complete Matrigel (see Recipes)
  37. 0.1 M phosphate buffered saline (PBS) (see Recipes)
  38. 4% paraformaldehyde (PFA) (see Recipes)
  39. PBS containing DAPI (see Recipes)
  40. Blocking buffer (see Recipes)
  41. Primary antibody (see Recipes)
  42. Secondary antibody (see Recipes)

Equipment

  1. Pipettes  
    20 µl pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4642050 )
    200 µl pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4642080 )
    1,000 µl pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4642090 )
  2. Biosafety cabinet, any brand/model
  3. Bottles, sterile glass or plastic, any brand, for tissue culture medium storage
  4. Millipore StericupTM sterile vacuum filter units (EMD Millipore, catalog number: SCGPU01RE )
  5. Dissection microscope (Olympus, or similar) with lamp (or cold light source)
  6. Dissecting scissors, 12.5 cm long, straight (Coherent Scientific, catalog number: 15922 )
  7. Iris scissors 10 cm long, 30° angle, supercut (Coherent Scientific, catalog number: 500046 )
  8. Dressing forceps, 15.5 cm long (Coherent Scientific, catalog number: 500363 )
  9. 2 x Dumont forceps #3, 12 cm long, 0.08 x 0.04 mm tips (Coherent Scientific, catalog number: 500337 )
  10. Dissecting spatula, 140 mm long, 3 wide mm blade (World Precision Instruments, catalog number: 501772 )
  11. Scalpel handle No. 3 with scalpel blade No. 15 (Coherent Scientific , catalog numbers: 500236 and 500242 )
  12. Dumont forceps #5, 11 cm long, 0.06 x 0.01 mm tips (Coherent Scientific, catalog number: 14095 )
  13. Humidified tissue culture incubator (5% CO2, 37 °C), any brand/model
  14. Refrigerator (4 °C), any brand/model
  15. Freezer (-20 °C), any brand/model
  16. Inverted fluorescent microscope and digital camera (Olympus, model: IX81 or similar)

Software

  1. Axiovision software v4.1 (Zeiss, Thornwood, NY) or similar
  2. GraphPad Prism (v4.03)

Procedure

  1. Prepare working area
    1. Disinfect equipment using 80% ethanol (see Recipe 1), and place into a biosafety cabinet: dissection tools, 35 mm and 10 cm culture plates, media bottles, serological pipettes (5 ml) and pipette-boy, ice tray and ice, barrier pipette tips and relevant pipettes, and dissection microscope.
    2. Label all required dishes and tubes appropriately.

  2. Brain dissection
    1. Decapitate postnatal 1-2 day old C57Bl/6 mouse pups using dissecting scissors (along green dotted line) (Figure 1A). Dispose of body into biohazard bag.
    2. Using iris scissors, cut the skin on the scalp mid-sagittally (purple arrow) and peel laterally to expose the skull. Using dressing forceps and iris scissors, cut away the excess skin (Figure 1B).
    3. Using iris scissors, make the same cut through the skull, and two perpendicular cuts (blue arrows; Figure 1B). Using Dumont forceps #3 peel back the skull between these cuts to expose the underlying brain.


      Figure 1. Step-by-step dissection procedure depicted in schematic and microscopic images. A-C. Removal of the brain from 1-2 day old mouse pups; D-F. Sectioning the rostral brain, dissection of RMS, and cutting RMS into many small pieces approximately 200-300 µm in diameter; G. Culturing up to 4 explants far apart in Matrigel with test compound(s) that are maintained in complete Neurobasal media. Bar represents 2 mm.

    4. Using the flat part of the spatula, starting at the caudal end of the brain, gently lift the brain out of the skull (Figure 1C). Place the brain into a 10 cm culture dish containing 5 ml HBSS media. Dispose of mouse head into biohazard bag.
    5. Using two pairs of Dumont forceps #3, gently pull the meninges off the brain, starting with the olfactory bulbs.

  3. Rostral migratory stream (RMS) dissection and explant production
    1. Using Dumont forceps #3 to hold the brain steady, use a scalpel blade at the rostral end of the brain to cut 3-4 coronal slices throughout the brain (Figure 1D). When using the scalpel blade, use a gentle back-and-forth sawing motion to preserve the morphology of the tissue.
    2. Using Dumont forceps #5, transfer rostral slices from both brain hemispheres containing RMS (which also contain migrating anterior subventricular zone [aSVZ] neuroblasts), into 35 mm dishes containing ice-cold HBSS (Figure 1E).
    3. To identify the RMS within these slices, angle the light source onto the center of the section so that the central RMS can be distinguished from the surrounding tissue. In the early postnatal mouse, the RMS will appear more translucent, dark and shiny than the surrounding tissue. The RMS should be located centrally, and at its widest point be about one third of the width of each coronal slice.
    4. Using two insulin syringe needles, dissect out the RMS by making cuts on the inside of its border (Figure 1E).
    5. Using the same syringe needles, cut the RMS into many small pieces approximately 200-300 µm wide, and set aside (Figure 1F).

    Notes:

    1. The authors did not use a tissue/brain slicer to cut the brain into 3 or 4 coronal slices, although this equipment could assist others to cut more straight and even slices throughout the brain.
    2. To better visualize the RMS within a coronal slice, it may be important to re-arrange the light source so that the RMS appears darker from the surrounding tissue and thus can be distinguished more easily.
    3. For better experimental analysis, use the needles to cut the RMS into pieces that are approximately the same size and shape.
    4. One RMS may produce up to 50 explants.

  4. Explant experimentation
    1. Using ice-cold pipette tips and on top of ice tray, prepare complete Matrigel (see Recipe 3) by diluting Matrigel in complete neurobasal medium (see Recipe 2). To create different treatment conditions, add a maximum of 2 µl of the compound(s) to be tested into the diluted Matrigel (Figure 1G). Store on ice.
    2. Place a 4-well dish inside a 10 cm dish filled with ice to enable longer manipulation time of explants before the Matrigel polymerizes.
    3. Preparing one well at a time, using Dumont forceps #5, carefully pick up an explant and place into one well of the 4-well dish. Plate four RMS explants per well, and remove excess HBSS using a 10 µl pipette.
    4. Turn down the light source brightness to reduce any heat output (or use a cold light source illuminator).
    5. Using a scalpel blade, trim the tip of an ice-cold pipette tip to make a wider opening, and transfer 100 µl of diluted Matrigel onto explants in one well. Using the same tip, gently mix the Matrigel with the explants (without making any air bubbles), ensuring explants are entirely surrounded by the gel. Work quickly during this step otherwise the gel will begin to polymerize as it warms up under the heat of the microscope lamp.
    6. Using Dumont forceps #3, arrange the explants in the Matrigel so that they are far apart from each other, and far apart from the walls of the well.
    7. Repeat for other wells.
    8. Place two 35 mm 4-well dishes (containing explants and Matrigel) in a 10 cm dish and incubate (5% CO2, 37 °C) for 15 min in order for the Matrigel to polymerize.
    9. After polymerization, gently add 2 ml of complete neurobasal media to cover Matrigel and explants.
    10. Maintain cultures in a humidified incubator (5% CO2, 37 °C) for 72 h.

    Notes:

    1. To prevent Matrigel from prematurely polymerizing, ensure manipulation is performed in ice-cold conditions using cell culture dishes and pipette tips pre-chilled at -20 °C.
    2. Mix the explants well in the Matrigel without making any bubbles, and position the explants in the wells far apart from each other, and away from the walls of the well to allow migration in all directions.
    3. Once the Matrigel and explants have been placed in the well it may become difficult to move the explants within the Matrigel. To assist with this process, prepare one well at a time. Between wells it may be necessary to turn off the light source (i.e., eliminate any heat sources) for 2-3 min and place a 10 cm Petri dish filled with ice on top of the dissecting microscope stage to cool it down quickly.
    4. Using a marker pen, keep track of the 4 explants in each well by labelling the bottom of the well 1, 2, 3 and 4.

  5. Explant immunohistochemistry
    1. Warm 4% PFA (see Recipe 5) to 37 °C
    2. Use a disposable transfer pipette to gently transfer solutions on and off the explants as follows. 
      1. Remove media and add 2 ml warm PFA. Incubate for 1 h at room temperature.
      2.  Remove PFA, and wash explants with PBS containing DAPI (see Recipe 6).
      3. Incubate explants in 2 ml blocking buffer (see Recipe 7) for 30 min at room temperature.
      4. Remove blocking buffer and incubate in 2 ml primary antibody solution (see Recipe 8) overnight at 4 °C.
      5. The next day gently wash the explants three times with PBS.
      6.  Incubate explants in 2 ml fluorescent secondary antibody (see Recipe 9) for 48 h at 4 °C.
      7. Gently wash explants again three times with PBS.
    3. Gently remove blocking buffer from wells.

Data analysis

  1. Explant imaging and analysis
    1. Photograph explants on an inverted fluorescent microscope at 4x magnification.
    2. Categorize neuroblast migration away from RMS explant into 3 groups, as follows:
      1     
      Explants that only give rise to compact chains
      2
      Explants that exhibit mixed cell outgrowth (individual and chains)
      3
      Explants that only exhibit cells migrating individually
       
    3. Grade the extent of chain and cell outgrowth using a semi-quantitative scale, as follows (Figures 2A-2E): 
      0 = No outgrowth
      1 = Less than 10 chains/cells
      2 = Approximately 10-50 chains/cells
      3 = Approximately 50-100 chains/cells
      4 = Extensive growth (greater than 100 chains/cells)


      Figure 2. Grading chain/cell outgrowth from explant using semi-quantitative scale from 0-4. A. Zero (0) represents no growth; B. One represents less than 10 chains/cells; C. Two represents ~10-50 chains/cells; D. Three represents ~50-100 chains/cells; E. Four represents extensive growth > 100 chains/cells. F-J. Outgrowth area is calculated by subtracting explant area from total growth area, and longest migration length is determined using a line drawing tool through the explant epicenter. Bar represents 0.2 mm.

    4. For each explant, measure the area of migration and longest migration length using Axiovision software v4.1 (Zeiss, Thornwood, NY), or similar Image Analysis software (Figures 2F-2J). As the explant sizes are not all the same, the area is measured for each explant by subtracting the area of the explant body from the total area of outgrowth (Figure 2H), and then normalize the area of outgrowth by expressing it as a ratio of outgrowth area:explant body area.
    5. Longest migration length can also be measured by using a line drawing tool as previously described (Cregg et al., 2010). First, the explant epicenter must be identify (yellow bars; Figure 2G), then a series of bars are drawn across the entire area of outgrowth intersecting the explant epicenter (blue bars; Figure 2I). The longest line from epicenter to outgrowth perimeter represents the longest migration length (Figure 2J).
    6. Average all results across different explant treatments, and then average results across individual experiments, and graph mean ± SEM. Assess data for homogeneity of variance using GraphPad Prism (v4.03). If a normal distribution is found, analyze data using one-way ANOVA, making individual comparisons with the Bonferroni post-hoc test. Set significance at P < 0.05.
    7. Perform analysis on at least 10-12 explants per treatment condition, and repeat experiment at least 3 times to create replicates.
    Note: Although there may be varying degrees of migration of neuroblasts exiting the explants, the authors include all explants in the analysis. Two exceptions are, if explants are sitting in the Matrigel at an angle and are thus not able to be imaged correctly, and/or if the explants are not completely submerged in the Matrigel with part of the tissue floating in the media.

Recipes

  1. 80% ethanol solution
    80 ml pure ethanol
    20 ml H2O
  2. Complete neurobasal medium
    50 ml neurobasal medium
    1 ml B27 (50x)
    1 ml glutamine (4 mM)
    500 µl penicillin/streptomycin (10,000 U/ml)
    0.5 µl bFGF (1 mg/ml)
    0.5 µl EGF (1 ng/ml)
    Filter media using sterile vacuum filter units and store in the bottles, or in 50 ml tissue culture sterile conical tubes
  3. Complete Matrigel
    3 ml growth factor reduced Matrigel
    1 ml complete neurobasal medium
    In advance, thaw Matrigel stock solution overnight at 4 °C and make 0.5 ml aliquots using cold pipette tips and cold Eppendorf tubes, then store at -20 °C. On the day of the explant assay, thaw individual tubes on ice and dilute in complete neurobasal medium
  4. 0.1 M phosphate buffered saline (PBS)
    16 mM Na2HPO4
    4 mM Na2HPO4·H2O
    150 mM NaCl
    Adjust to pH 7.4
  5. 4% paraformaldehyde (PFA)
    4 g paraformaldehyde
    Make up to 100 ml with PBS
    Heat to a maximum of 60 °C
    Add a few drops of NaOH until the solution turns clear
    Adjust to pH 7.4
  6. PBS containing DAPI
    10 ml PBS
    1 µl DAPI
  7. Blocking buffer
    5 ml donkey serum (5%, v/v)
    0.5 ml Triton X-100 (0.5%, v/v)
    Make up to 100 ml with PBS
  8. Primary antibody
    2 µl Goat anti-doublecortin (DCX) antibody
    1 ml blocking buffer
  9. Secondary antibody
    2 µl Cy3-conjugated donkey anti-goat
    1 ml PBS

Acknowledgments

Studies were supported by NIH/NINDS (DJL: NS049545 and NS30291) and an NH&MRC fellowship (AMT: 628344). The work was adapted from Dixon et al. (2016).

References

  1. Cregg, J. M., Wiseman, S. L., Pietrzak-Goetze, M. N., Smith, M. R., Jaroch, D. B., Clupper, D. C. and Gilbert, R. J. (2010). A rapid, quantitiative method for assessing axonal extension on biomaterial platforms. Tissue Eng Part C Methods 16(2):167-172.
  2. Dixon, K. J., Mier, J., Gajavelli, S., Turbic, A., Bullock, R., Turnley, A. M. and Liebl, D. J. (2016). EphrinB3 restricts endogenous neural stem cell migration after traumatic brain injury. Stem Cell Res 17(3): 504-513.
  3. Dixon, K. J., Theus, M. H., Nelersa, C. M., Mier, J., Travieso, L. G., Yu, T. S., Kernie, S. G. and Liebl, D. J. (2015). Endogenous neural stem/progenitor cells stabilize the cortical microenvironment after traumatic brain injury. J Neurotrauma 32(11): 753-764.
  4. Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1999). Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci U S A 96(20): 11619-11624.
  5. Garcia-Verdugo, J. M., Doetsch, F., Wichterle, H., Lim, D. A. and Alvarez-Buylla, A. (1998). Architecture and cell types of the adult subventricular zone: in search of the stem cells. J Neurobiol 36(2): 234-248.
  6. Leong, S. Y., Faux, C. H., Turbic, A., Dixon, K. J. and Turnley, A. M. (2011). The Rho kinase pathway regulates mouse adult neural precursor cell migration. Stem Cells 29(2): 332-343.
  7. Li, B., Piao, C. S., Liu, X. Y., Guo, W. P., Xue, Y. Q., Duan, W. M., Gonzalez-Toledo, M. E. and Zhao, L. R. (2010). Brain self-protection: the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia. Brain Res 1327: 91-102.
  8. Lois, C. and Alvarez-Buylla, A. (1994). Long-distance neuronal migration in the adult mammalian brain. Science 264(5162): 1145-1148.
  9. Mirzadeh, Z., Doetsch, F., Sawamoto, K., Wichterle, H. and Alvarez-Buylla, A. (2010). The subventricular zone en-face: wholemount staining and ependymal flow. J Vis Exp (39).
  10. Paez-Gonzalez, P., Asrican, B., Rodriguez, E. and Kuo, C. T. (2014). Identification of distinct ChAT(+) neurons and activity-dependent control of postnatal SVZ neurogenesis. Nat Neurosci 17(7): 934-942.
  11. Ramaswamy, S., Goings, G. E., Soderstrom, K. E., Szele, F. G. and Kozlowski, D. A. (2005). Cellular proliferation and migration following a controlled cortical impact in the mouse. Brain Res 1053(1-2): 38-53.
  12. Reynolds, B. A. and Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255(5052): 1707-1710.
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简介

哺乳动物前脑中的室下区(SVZ)含有在整个成年期间通过流行性流(RMS)迁移到嗅球的茎/祖细胞。 SVZ衍生的外植体培养提供了一种方便的方法来评估调节神经干/祖细胞迁移的中间阶段的因素。在这里,我们描述了SVZ衍生的RMS外植体从新生儿小鼠脑中的分离以及培养和评估其迁移所需的条件。


背景 成年哺乳动物前脑含有一个位于啮齿动物和人类侧脑室旁边的神经源性小生境,并被恰当地命名为室下区(SVZ)。在啮齿动物中,SVZ是细胞的薄的“楔形”,覆盖了侧脑室的整个壁(Mirzadeh等人,2010; Paez-Gonzalez等人。 ,2014; Dixon等人,2016)。在SVZ内,慢分化的星形胶质细胞样B细胞分化成快速分裂的C型神经祖细胞(也称为转运扩增细胞),其产生双皮层蛋白阳性的A型神经母细胞,尽管少突胶质细胞和星形胶质细胞也能够(Garcia-Verdugo等人,1998; Tavazoie等人,2008; Rikani等人,2013)。在啮齿动物中,每天估计有10,000到30,000个神经母细胞。这些成神经细胞在通过流行流体流(RMS)迁移到嗅球时形成链(Lois和Alvarez-Buylla,1994; Sun等人,2010)。消融研究表明,快速分裂型C神经祖细胞需要大约2天才能填充SVZ,另外需要2.5天的成神经细胞出现(Doetsch等人,1999)。这些成神经细胞中的一小部分能够异位移出RMS到周围组织的初始小鼠中;然而,这种现象在脑损伤后急剧增加(Dixon等人,2016)。神经母细胞将其迁移路线重定向到受损组织的能力已被证明对脑恢复​​具有有益的影响(Li等人,2010; Dixon等人,2015, ),其可以在损伤后3天发生(Ramaswamy等人,2005; Dixon等人,2016)。
&NBSP; 1992年雷诺兹和魏斯(Reynolds and Weiss,1992)首次确定了文化干细胞的自我更新能力。在胰蛋白酶消化,解离和培养之前,作者使用精细解剖来收获小块成年小鼠纹状体。这种原始方案和随后的变化现在被广泛用于生长神经球或单层培养物,以评估调节干细胞存活,增殖和/或分化成神经元的因子(Theus等人,2012)。这些培养系统依赖于生长因子(即成纤维细胞和表皮生长因子)的存在来维持增殖状态,而这些因子的撤出诱导快速分化成成熟神经元。不幸的是,这些条件限制了分析调节A型神经母细胞的因素的能力,这是干细胞和神经元之间的一个暂时的阶段。抵消这个限制;可将SVZ衍生的组织收获并培养成含有基质胶质的层粘连蛋白和胶原蛋白中的外植体,其将神经干细胞维持在其成神经细胞状态,使其迁移(Ward和Rao,2005; Dixon等人,2016)。此外,培养的SVZ外植体的成神经细胞迁移具有与RMS中观察到的特征相似的特征。在这里,我们描述了从Ward和同事(Leong等人,2011)修改的RMS外植体方法,用于研究SVZ衍生的成神经细胞的链移动。

关键字:神经母细胞, 神经干细胞, SVZ源性外植体, 嘴侧迁移流, 基质胶, 体外

材料和试剂

  1. 35毫米细胞培养皿与4内孔,每个直径为10毫米(Greiner Bio One国际,目录号:627170)
  2. 10厘米细胞培养皿(康宁,目录号:353003)
  3. 预冷(-20°C)组织培养移液管过滤嘴
  4. 10μl提示(Corning,Axygen ®,目录号:TF-300-R-S)
  5. 200μl提示(Corning,Axygen ®,目录号:TF-200-R-S)
  6. 1000μl提示(Corning,Axygen ®,目录号:TF-1000-R-S)
  7. 5 ml血清移液管和移液器男孩(VWR,目录号:612-3702)
  8. 1.5ml Eppendorf微量离心管(VWR,目录号:211-0007)
  9. 3.2 ml一次性移液器(Thermo Fisher Scientific,目录号:BER202-1S)
  10. 1 ml带有可拆卸针头的胰岛素注射器(BD,目录号:329651)
  11. 冰盘和冰可用
  12. 小生物危害袋可用
  13. 用于标记细胞培养板的标记笔(例如,,Sharpie)
  14. 1-2日出生日C57Bl/6野生型小鼠(澳大利亚动物资源中心)或当地动物供应商
  15. 绝对乙醇(Sigma-Aldrich,目录号:24102)
  16. Hanks平衡盐溶液(HBSS)(Thermo Fisher Sientific,Gibco TM,目录号:14170112)
  17. 生长因子减少Matrigel(康宁,目录号:356230)
  18. Neurobasal培养基(Thermo Fisher Sientific,Gibco TM,目录号:21103049)
  19. B27补充x50(Thermo Fisher Sientific,Gibco TM ,目录号:17504044)
  20. 200mM谷氨酰胺(Thermo Fisher Sientific,Gibco TM,目录号:25030081)
  21. 青霉素/链霉素(10,000U/ml)(Thermo Fisher Sientific,Gibco TM,目录号:15140122)
  22. 重组鼠成纤维细胞生长因子(FGF)碱性1mg/ml(PeproTech,目录号:450-33)
  23. 重组鼠表皮生长因子(EGF)1mg/ml(PeproTech,目录号:315-09)
  24. 多聚甲醛(Sigma-Aldrich,目录号:158127)
  25. 磷酸氢二钠(Na 2 HPO 4)(Chem Supply,目录号:SA026)
  26. 磷酸二氢钠一水合物(NaH 2 PO 4·H 2 O)(Chem Supply,目录号:SO03310500)
  27. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  28. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S5881)
  29. DAPI(Thermo Fisher Sientific,Gibco TM ,目录号:D1306)
  30. 驴血清(Sigma Aldrich,目录号:D9663)
  31. Triton X-100(Sigma-Aldrich,目录号:X100)
  32. 山羊抗双皮质素(DCX)抗体(Santa Cruz Biotechnology,目录号:sc-8066或sc-271390)
    注意:作者使用sc-8066,该抗体已被停用。来自Santa Cruz Biotechnology的建议替代品是sc-271390。
  33. Cy3缀合的驴抗山羊抗体(Jackson ImmunoResearch,目录号:705-165-147)
  34. 80%乙醇溶液(参见食谱)
  35. 完成神经巴氏培养基(见食谱)
  36. 完整Matrigel(见食谱)
  37. 0.1M磷酸缓冲盐水(PBS)(参见食谱)
  38. 4%多聚甲醛(PFA)(见配方)
  39. 含有DAPI的PBS(参见食谱)
  40. 阻塞缓冲区(见配方)
  41. 一抗(见食谱)
  42. 二抗(见食谱)

设备

  1. 移液器
    20微升移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4642050)
    200μl移液器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:4642080)
    1,000μl移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4642090)
  2. 生物安全柜,任何品牌/型号
  3. 瓶,无菌玻璃或塑料,任何品牌,用于组织培养基储存
  4. Millipore Stericup TM无菌真空过滤器(EMD Millipore,目录号:SCGPU01RE)
  5. 带有灯(或冷光源)的解剖显微镜(Olympus或类似物)
  6. 解剖剪刀,12.5厘米长,直(Coherent Scientific,目录号:15922)
  7. 虹膜剪刀10厘米长,30度角,超切(Coherent Scientific,目录号:500046)
  8. 敷料钳,15.5厘米长(相干科学,目录号:500363)
  9. 2 x杜蒙镊子#3,12厘米长,0.08 x 0.04毫米尖(Coherent Scientific,目录号:500337)
  10. 解剖刮刀,140毫米长,3宽mm刀片(世界精密仪器,目录号:501772)
  11. 手术刀第15号手术刀第3号(Coherent Scientific,目录号:500236和500242)
  12. Dumont镊子#5,11厘米长,0.06 x 0.01毫米提示(Coherent Scientific,目录号:14095)
  13. 加湿组织培养箱(5%CO 2,37℃),任何品牌/型号
  14. 冰箱(4°C),任何品牌/型号
  15. 冰柜(-20°C),任何品牌/型号
  16. 倒置荧光显微镜和数码相机(Olympus,型号:IX81或类似)

软件

  1. Axiovision软件v4.1(Zeiss,Thornwood,NY)或类似的
  2. GraphPad Prism(v4.03)

程序

  1. 准备工作区
    1. 使用80%乙醇消毒设备(参见食谱1),并放入生物安全柜:夹层工具,35 mm和10 cm培养板,培养瓶,血清移液管(5ml)和移液器 - 男孩,冰盘和冰,屏障移液器吸头和相关移液器和解剖显微镜。
    2. 适当地标记所有必需的菜肴和管子。

  2. 脑解剖
    1. 使用解剖剪刀(沿着绿色的虚线),使产后1-2天龄的C57Bl/6小鼠幼仔蜕皮(图1A)。将身体放入生物危害袋。
    2. 使用虹膜剪刀剪下头皮上的皮肤(紫色箭头),并横向剥离暴露头骨。使用敷料钳和虹膜剪刀切掉多余的皮肤(图1B)
    3. 使用虹膜剪刀,通过颅骨进行相同的切割,并且进行两个垂直切割(蓝色箭头;图1B)。使用Dumont镊子#3剥离这些切口之间的头骨,以暴露下面的大脑。


      图1.在示意图和显微镜图像中描述的逐步解剖程序。 A-C。从1-2天老鼠小鼠中去除大脑; D-F。切分大脑,解剖RMS,并将RMS切割成直径约200-300μm的许多小块; G.在Matrigel中培养多达4个外植体,其中测试化合物保持在完整的Neruobasal培养基中。酒吧代表2毫米。

    4. 使用刮刀的平坦部分,从大脑的尾端开始,轻轻地将脑部从头骨中提出(图1C)。将大脑放入含有5ml HBSS培养基的10cm培养皿中。将鼠标头放入生物危险袋中。
    5. 使用两对Dumont镊子#3,从嗅球开始轻轻地将脑膜从脑部拉出。

  3. 传播流(RMS)解剖和外植体生产
    1. 使用Dumont镊子#3来保持大脑稳定,使用大脑头端的解剖刀刀片切割整个大脑中的3-4个冠状切片(图1D)。当使用手术刀刀片时,使用温和的来回锯切运动来保持组织的形态。
    2. 使用Dumont镊子#5,将含有RMS(其也包括迁移的前室下区[aSVZ]成神经细胞)的两个脑半球的切片转移到含有冰冷HBSS的35mm培养皿中(图1E)。
    3. 为了识别这些切片内的均方根,将光源定位在部分的中心,以使中心RMS与周围组织区分开。在早期出生后的鼠标中,RMS会比周围的组织显得更加半透明,黑暗和光泽。 RMS应位于中心位置,其最宽点约为冠状切片宽度的三分之一。
    4. 使用两只胰岛素注射器针头,通过在其边界的内侧切割RMS来解剖RMS(图1E)
    5. 使用相同的注射器针头,将RMS切割成大约200-300μm宽的许多小件,并放在一边(图1F)。

    注意:

    1. 作者没有使用组织/脑切片机将大脑切成3或4个冠状切片,尽管这种设备可以帮助其他人在整个大脑中切割更平直甚至切片。
    2. 为了更好地可视化冠状切片中的RMS,重新排列光源可能很重要,以使RMS从周围组织变得更暗,因此可以更容易地区分。
    3. 为了更好的实验分析,使用针将RMS切割成与大小和形状大致相同的片段。
    4. 一个RMS可能产生多达50个外植体。

  4. 外部实验
    1. 使用冰冷的移液管吸头和冰盘顶部,通过在完整的神经巴氏培养基中稀释Matrigel来制备完整的Matrigel(参见食谱3)(参见方法2)。为了产生不同的处理条件,最多可将2μl待测化合物加入稀释的基质胶中(图1G)。在冰上存放
    2. 在装满冰的10厘米盘子中放置一个4孔盘,使Matrigel聚合之前的外植体操作时间更长。
    3. 一次准备好一个,使用Dumont镊子#5,仔细拿起外植体,放入4孔盘的一口。每孔平板四个RMS外植体,并使用10μl移液管除去过量的HBSS
    4. 调低光源亮度以减少任何热量输出(或使用冷光源照明器)。
    5. 使用手术刀刀片,修剪冰冷的移液管尖端,使开口更宽,并将100μl稀释的基质胶转移到一个孔中的外植体上。使用相同的尖端,轻轻地将基质胶与外植体混合(不产生任何气泡),确保外植体完全被凝胶包围。在此步骤中快速工作,否则凝胶将在显微镜灯的加热下开始聚合。
    6. 使用Dumont镊子#3,将外植体排列在Matrigel中,使它们彼此分开,并远离井壁。
    7. 对其他井重复。
    8. 将两个35毫米4孔的培养皿(含外植体和Matrigel)放在10厘米的培养皿中并孵育(5%CO 2,37℃)15分钟,以使Matrigel聚合。 br />
    9. 聚合后,轻轻加入2毫升完整的神经细胞培养基,以覆盖Matrigel和外植体
    10. 在潮湿的培养箱(5%CO 2,37℃)中维持培养72小时。

    注意:

    1. 为了防止Matrigel过早聚合,请确保在冰冷条件下使用细胞培养皿和在-20°C预冷的移液管吸头进行操作。
    2. 将外植体在Matrigel中很好地混合,而不会产生任何气泡,并将外植体放置在彼此远离的井中,并远离井壁,以允许在所有方向迁移。 >
    3. 一旦Matrigel和外植体已经放入井中,可能会难以移动Matrigel中的外植体。为了协助这个过程,一次准备好一口井。在井之间,可能需要关闭光源(即,消除任何热源)2-3分钟,并在解剖显微镜载物台的顶部放置装满冰的10厘米培养皿,以快速冷却。 em>
    4. 使用标记笔,通过标注井1,2,3和4的底部来跟踪每个井中的4个外植体。

  5. 外植体免疫组织化学
    1. 温热4%PFA(见配方5)至37°C
    2. 使用一次性移液移液器轻轻地将解决方案转移到外植体上,如下所示。
      1. 取出培养基并加入2ml温热的PFA。在室温下孵育1小时。
      2.  去除PFA,并用含有DAPI的PBS洗涤外植体(参见配方6)。
      3. 将外植体在2ml封闭缓冲液(见方案7)中孵育30分钟。
      4. 去除封闭缓冲液,并在4℃下在2ml一抗溶液(见方案8)中孵育过夜。
      5. 第二天用PBS轻轻洗涤外植体三次。
      6. 在4℃下将外植体孵育2毫升荧光二抗(参见食谱9)48小时。
      7. 用PBS轻轻洗涤外植体三次。
    3. 轻轻取出孔中的封闭缓冲液。

数据分析

  1. 外显成像与分析
    1. 以4倍放大倍数的倒置荧光显微镜照片外植体
    2. 将神经母细胞迁移从RMS外植体分为3组,如下:
      1    
      只能引起紧凑型链条的外展
      2
      展示混合细胞生长(个体和链)的外植体
      3
      仅显示单独迁移的细胞的外显子
       
    3. 如下(图2A-2E),使用半定量尺度对链和细胞生长的程度进行分级:(a)
      0 =不成长
      1 =小于10个链/细胞
      2 =大约10-50个链/细胞
      3 =大约50-100条链/细胞
      4 =广泛的增长(大于100个链/细胞)


      图2.使用半定量量表从外植体分级链/细胞生长从0-4。 A.零(0)表示无生长;一个代表小于10个链/细胞;两个代表〜10-50个链/细胞; D.三代〜50-100条链/细胞;四代表广泛的成长> 100条链/细胞。 F-学家通过从总生长区域减去外植体面积计算出生长面积,并且通过外植体中心使用线描工具确定最长的迁移长度。酒吧代表0.2毫米。

    4. 对于每个外植体,使用Axiovision软件v4.1(Zeiss,Thornwood,NY)或类似的图像分析软件(图2F-2J)测量迁移面积和最长的迁移长度。由于外植体大小不尽相同,所以通过从外生长的总面积减去外植体的面积来测量每个外植体的面积(图2H),然后通过将外植体的面积表示为生长区:外植体。
    5. 也可以通过使用如上所述的线绘图工具(Cregg等人,2010)来测量最长的迁移长度。首先,外植体中心必须识别(黄色条;图2G),然后将一系列条绘制在与外植体震中相交的整个生长区域(蓝条;图2I)。从震中到长周长的最长线代表最长的移动长度(图2J)
    6. 平均所有结果跨不同的外植体治疗,然后平均结果跨个别实验,和图表平均值±SEM。使用GraphPad Prism(v4.03)评估方差均匀性的数据。如果发现正常分布,使用单因素方差分析来分析数据,与Bonferroni事后检测进行单独比较。在 P 0.05。
    7. 对每个治疗条件至少10-12个外植体进行分析,并重复实验至少3次以创建重复。
    注意:虽然离开外植体的成神经细胞可能有不同程度的迁移,但作者包括分析中的所有外植体。两个例外是,如果外植体以某种角度坐在Matrigel中,因此不能正确成像,和/或如果外植体没有完全浸没在Matrigel中,部分组织漂浮在培养基中。

食谱

  1. 80%乙醇溶液
    80毫升纯乙醇
    20ml H 2 O O
  2. 完成神经支气管介质
    50毫升神经巴氏培养基
    1 ml B27(50x)
    1ml谷氨酰胺(4mM)
    500μl青霉素/链霉素(10,000 U/ml)
    0.5μlbFGF(1 mg/ml)
    0.5μlEGF(1 ng/ml)
    过滤介质使用无菌真空过滤器单元并存储在瓶子中,或在50ml组织培养无菌锥形管中
  3. 完整Matrigel
    3 ml生长因子降低Matrigel
    1毫升完整的神经巴氏培养基 提前,将Matrigel储存溶液在4℃下溶解过夜,并使用冷移液管吸头和冷的Eppendorf管制备0.5ml等分试样,然后储存在-20°C。在外植体测定当天,在冰上解冻各个管,并在完整的神经巴氏培养基中稀释
  4. 0.1M磷酸缓冲盐水(PBS)
    16mM Na 2 HPO 4
    4mM Na 2 HPO 4 H 2 O 2// 150 mM NaCl
    调整至pH 7.4
  5. 4%多聚甲醛(PFA)
    4克多聚甲醛
    用PBS
    达到100ml 加热至最高60°C
    加入几滴NaOH直至溶液变清净 调整至pH 7.4
  6. 含有DAPI的PBS
    10ml PBS
    1μlDAPI
  7. 阻塞缓冲区
    5毫升驴血清(5%,v/v)
    0.5ml Triton X-100(0.5%,v/v)
    用PBS
    达到100ml
  8. 初级抗体
    2μl山羊抗双皮质素(DCX)抗体
    1 ml阻塞缓冲液
  9. 二次抗体
    2μlCy3缀合的驴抗山羊
    1 ml PBS

致谢

研究由NIH/NINDS(DJL:NS049545和NS30291)和NH& MRC研究金(AMT:628344)支持。这项工作改编自Dixon等人。(2016)。

参考

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  2. Dixon,KJ,Mier,J.,Gajavelli,S.,Turbic,A.,Bullock,R.,Turnley,AM and Liebl,DJ(2016)。  EphrinB3限制创伤性脑损伤后的内源性神经干细胞迁移。 干细胞研究(3):504-513。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dixon, K. J., Turbic, A., Turnley, A. M. and Liebl, D. J. (2017). Explant Methodology for Analyzing Neuroblast Migration. Bio-protocol 7(9): e2249. DOI: 10.21769/BioProtoc.2249.
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