搜索

Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina
采用异时性细胞团分析法检测小鼠视网膜中的细胞间通讯   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.

Keywords: Heterochronic pellet assay(异时性细胞团分析法), Retinal differentiation(视网膜分化), Retinal progenitor cells(视网膜祖细胞), Re-aggregation(重新聚合), Amacrine cell(无长突细胞), Negative feedback signaling(负反馈信号), Heterotypic cell interactions(异型细胞相互作用)

Background

Several mechanisms are employed to ensure that the correct numbers of differentiated cells are generated during organ and tissue development. For example, progenitor cells may respond to the levels of hormones or growth factors secreted by differentiated cells, progenitors may count the number of divisions they undergo, or there may be a mechanism to count the final number of differentiated cells (Lui and Baron, 2011). In the retina, negative feedback signals that are secreted by differentiated cells are sensed by progenitor cells, which stop producing that differentiated cell type when the signals reach threshold levels (Belliveau and Cepko, 1999; Reh and Tully, 1986; Waid and McLoon, 1998). We and other have demonstrated that Shh is an essential negative regulator of a RGC fate (Wang et al., 2005; Zhang and Yang, 2001). We also dissected the feedback process for retinal amacrine cells, showing that the transcription factor Zac1 acts in amacrine cells to initiate transforming growth factor b2 (Tgfb2) expression, which negatively regulates RPC proliferation and amacrine cell differentiation (Ma et al., 2007). Notably, other TGFβ family members have similar feedback functions in the olfactory epithelium (Wu et al., 2003), pancreas (Harmon et al., 2004), and skeletal muscle (Tobin and Celeste, 2005). We also used the heterochronic pellet assay to examine how amacrine cell feedback signals are themselves regulated. We found that Pten is an essential positive regulator of amacrine cell differentiation, and using the pellet assay, we demonstrated that Pten acts in RPCs to control responsiveness to Tgfβ2 signaling (Tachibana et al., 2016). Understanding how amacrine cells and RPCs interact provides important new insights into how cell number is controlled in the retina. Notably, similar interactions between Pten and Tgfβ signaling may underlie cell number control in other vertebrate organs where Tgfβ signaling is an important determinant of organ size.

Materials and Reagents

  1. Fisherbrand sterile 100 x 15 mm polystyrene Petri dish (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875713 )
  2. 15 ml conical tubes (SARSTEDT, catalog number: 62.554.502 )
  3. Sterile individually packaged 5 ml pipettes (SARSTEDT, catalog number: 86.1253.001 )
  4. Tissue culture 24-well plates (SARSTEDT, catalog number: 83.3922.300 )
  5. SamcoTM extra long transfer pipet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 262 )
  6. 13 mm, 0.8 μm Nuclepore Track-Etch membrane (GE Healthcare, catalog number: 110409 )
  7. Kimwipes (Kimberly-Clark Worldwide, catalog number: 34120 ) (not autoclaved, but kept clean)
  8. Superfrost Plus Micro slides (VWR, catalog number: 48311-703 )
  9. Standard microscope slide box (Heathrow Scientific, catalog number: HEA15991A )
  10. Micro cover glass (VWR, catalog number: 48404-454 )
  11. 4 in x 250 ft Parafilm roll (Bemis, catalog number: PM999 or VWR, catalog number: 52858-032 )
  12. 50 ml corning tube (SARSTEDT, catalog number: 62.547.254 )
  13. 0.22 μm sterilize filter filtropur (SARSTEDT, catalog number: 83.1826.001 )
  14. 60 ml syringe (Medtronic, catalog number: 8881560125 )
  15. Rapid-FlowTM sterile disposable bottle top filters (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 291-4520 )
  16. E15.5 CD1 mouse (Charles River Laboratories International, catalog number: 022 ) (see Note 1)
  17. P2 Ptenfl/fl; Pax6-Cre+ or Pten+/+ mouse (see Note 2)
  18. 1x Ca2+/Mg2+-free DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
  19. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  20. Aqua-Poly/Mount (Polysciences, catalog number: 18606 )
  21. Trypsin (Sigma-Aldrich, catalog number: T1005 )
  22. Heat inactivated FBS (fetal bovine serum) (Thermo Fisher Scientific, GibcoTM, catalog number: 12484028 )
  23. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  24. 1x HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
  25. Heat inactivated horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050088 )
  26. 200 mM L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  27. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  28. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  29. Amphotericin B (Thermo Fisher Scientific, GibcoTM, catalog number: 15290026 )
  30. 5-Bromouridine (BrdU) (Sigma-Aldrich, catalog number: 850187 )
  31. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  32. Potassium chloride (KCl) (EMD Millipore, catalog number: PX1405 )
  33. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
  34. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  35. Crystalline PFA (Sigma-Aldrich, catalog number: P6148 )
  36. Sucrose (Sigma-Aldrich, catalog number: S9378 )
  37. 11.8 M hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  38. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  39. 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) as pre-prepared working solution that is used as described by the manufacturer (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
  40. Antibodies against Pax6 and BrdU (see Recipes, Table 1)
    Pax6 (BioLegend, catalog number: 901301 )
    BrdU (Bio-Rad Laboratories, catalog number: OBT0030 )
    Donkey Anti-rabbit Alexa-Fluor 488 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21206 )
    Goat Anti-rat Alexa Fluor 568 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11077 )
  41. Optimal cutting temperature compound (VWR, catalog number: 95057-838 )
  42. 2.5% trypsin (see Recipes)
  43. 0.125% trypsin (see Recipes)
  44. Retinal explant media (REM) (see Recipes)
  45. 1 mg/ml BrdU (see Recipes)
  46. 10x phosphate-buffered saline (PBS) (see Recipes)
  47. 1x phosphate-buffered saline (PBS) (See Recipes)
  48. 20% paraformaldehyde (PFA) (see Recipes)
  49. 4% paraformaldehyde (PFA) (See Recipes)
  50. 20% sucrose (see Recipes)
  51. 2 N hydrochloric acid (see Recipes)
  52. 1x phosphate-buffered saline/0.1% Triton X-100 (PBT) (see Recipes)
  53. Blocking solution (see Recipes)

Equipment

  1. Dumont forceps #5 (Fine Science Tools, catalog number: 11252-20 )
  2. Dumont forceps #55 (Fine Science Tools, catalog number: 11255-20 )
  3. Dumont forceps AA (Fine Science Tools, catalog number: 11210-20 )
  4. Shallow form shaking water bath (Precision Scientific, catalog number: 66799 )
  5. Pipette pump (SP Scienceware - Bell-Art Products - H-B Instrument, catalog number: F37898-0000 )
  6. 37 °C, 5% CO2 water jacketed incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3110 )
  7. Refrigerated tabletop centrifuge for 15 ml conical tubes (Eppendorf, model: 5810 R )
  8. Hemacytometer chamber (Hausser Scientific, catalog number: 3100 )
  9. P20 pipetmen (Gilson, catalog number: F123600 )
  10. P200 pipetmen (Gilson, catalog number: F123601 )
  11. P1000 pipetmen (Gilson, catalog number: F123602 )
  12. Cryostat (Leica Biosystems, model: CM3050 S )
  13. -20 °C freezer
  14. Upright fluorescence microscope (Leica Microsystems, model: DM RXA2 )
  15. Autoclave
  16. 1 L beaker (Corning, Pyrex®, catalog number: 1395-1L )
  17. 500 ml beaker (Corning, Pyrex®, catalog number: 1395-500 )
  18. 250 ml Erlenmeyer flask (Corning, Pyrex®, catalog number: 4450-250 )
  19. Fume hood
  20. Stereomicroscope for dissection (Leica Microsystems, model: MZ6 )
  21. Inverted light microscope (Leica Microsystems, model: DMIL LED )

Software

  1. GraphPad Prism (GraphPad Software)

Procedure

Notes:

  1. Pre-warm 0.125% trypsin and REM (see Recipes) to 37 °C prior to the experiment.
  2. This protocol was performed without sterile technique, but we took extreme caution to keep the cells and equipment as clean as possible.
  1. Dissociating E15.5 mouse retina
    1. Enucleate the eyes from E15.5 CD1 embryos at room temperature (see Note 1 about mice).
    2. Place eyes in Petri dish and add cold 1x DPBS to cover (Figure 1A).


      Figure 1. Representative images of retina/retinal cells at different time points of the protocol. A. E15.5 eyes after enucleation; B. E15.5 retina after RPE removal; C. E15.5 retina sunk to the bottom of a 15 ml Corning tube; D. Trypsinizing the retinas in 37 °C water bath for 10 min; E. Dissociated retinal cells. F. Heterochronic retinal cell aggregate pelleted on the bottom of the 15 ml Corning tube (arrowhead); G. Cell pellet placed on the membrane (outlined by red dotted line); H. Cell pellet on membrane placed on the surface of REM (side view); I. Section of aggregate labelled with BrdU (red) and amacrine cell marker Pax6 (green). le: lens, re: retina, RPE: retinal pigment epithelium.

    3. Remove retinal pigment epithelium (RPE) and lens from eyes using Dumont forceps #55 and #5 (Figure 1B) (see Note 4). To remove RPE, grab a small area of the sclera with Dumont forceps #55, then stab onto the sclera with Dumont forceps #5 to cause incision. Grind the edge of the incision with both forceps, and slowly rip open the incision. Keep opening up the incision until sclera, cornea and optic nerve are removed. RPE should detach from retina along with cornea (Video 1). Keeping the eyes in cold 1x DPBS aids RPE detachment. Detach ciliary margin from the retina using the forceps. This loosens the lens attachment on the retina. Gently pull the lens out (Video 2). Replace 1x DPBS for every eye dissection to keep Petri dish and buffer cold.

      Video 1. Removing RPE from eye

      Video 2. Removing lens from eye

    4. Transfer retinas into 15 ml Corning tube (with 10 ml of 1x DPBS at room temperature) (Figure 1C). Allow retinas to sink to bottom of the tube. (We recommend collecting ~20 retinas [2 eyes from each of 10 embryos] for this protocol. This will give approximately 8.0 x 106 cells.)
    5. Remove as much of the 1x DPBS as possible using a 5 ml pipet. Make sure not to pipet out the retinal tissue.
    6. Add 100 μl of 0.125% trypsin (pre-warmed to 37 °C) to retinas, and incubate in 37 °C water bath for 10 min (Figure 1D). Please note that retina will not break apart on its own. Trituration is required to break cell-cell interactions.
    7. Add 5 ml of 20% FBS/1x DPBS to stop trypsinization.
    8. Homogenize retinal cells completely by pipetting up and down several times (i.e., at least 30 times) using a 5 ml pipet (i.e., triturate; Figure 1E) until no clusters of cells are observed macroscopically. Make sure to triturate gently to avoid any air bubbles as bubbles can damage cells. We recommend using a Pipette pump to make sure trituration is gentle. (Video 3) (see Note 5)

      Video 3. Triturating trypsinized retinal cells

    9. Spin down cells at 520 x g for 5 min, then remove as much solution as possible.
    10. Re-suspend cells in 1 ml of REM (pre-warmed to 37 °C).
    11. Add 3 µl of 1 mg/ml BrdU into cell resuspension. Vortex gently and briefly to mix.
    12. Incubate cell resuspension for 1 h at 37 °C, 5% CO2 in water jacketed incubator.

  2. Dissociating P2 mouse retina
    1. Meanwhile, dissect P2 mouse retinas by enucleating the eyes, and removing RPE and lens as described above (see Note 2 and Note 3 about mice).
    2. Transfer retinas into 15 ml Corning tube (with 10 ml of 1x DPBS at room temperature), keeping the two retinas from each mouse together in a separate tube as the genotype of each mouse is different. Allow retinas to sink to tube bottoms.
    3. Remove as much 1x DPBS as possible, then add 100 µl of 0.125% trypsin, and incubate in 37 °C water bath for 10 min.
    4. Stop the reaction by adding 5 ml of 20% FBS/1x DPBS, and triturate the cells by pipetting gently several times with 5 ml pipet and pipette pump.
    5. Spin down at 520 x g for 5 min, and remove solution as much as possible.
    6. Re-suspend the cells with 1 ml REM (pre-warmed to 37 °C).

  3. Preparation of heterochronic cell pellet
    1. Remove E15.5 CD1 dissociated cells from the incubator after 1 h incubation in BrdU.
    2. Spin down cells at 520 x g for 5 min, remove media as much as possible, then re-suspend in 1 ml REM. Repeat this step twice more to completely remove any trace of BrdU. Re-suspend the cells in 1 ml REM (pre-warmed to 37 °C).
    3. For both E15.5 and P2 cells, take 10 µl of the cell resuspension and mix with 10 µl trypan blue. Calculate the concentration of each cell resuspension using hemacytometer (calculation described in Figure 2). Make sure that the cells are dissociated completely (i.e., no clumps of cells or doublets). If you see any clumps, we recommend to re-dissociate the cells.


      Figure 2. Formula for calculating concentration of cell resuspension using hemacytometer. Note that the dilution factor is 2 as cell resuspension is diluted with trypan blue (10 μl + 10 μl).

    4. Dilute E15.5 cell resuspension to 1 x 106 cells/ml in REM. Aliquot 1 x 106 cells (1 ml) into 15 ml Corning tubes as a control. Re-dilute E15.5 cell resuspension to 0.5 x 106 cells/ml.
    5. Dilute P2 cell resuspension to 1.0 x 106 cells/ml in REM. Aliquot 1 x 106 cell (1 ml) into 15 ml Corning tubes. Add 100 µl (5.0 x 104 cells) of 0.5 x 106 cells/ml from E15.5 CD1 cell resuspension. Mix thoroughly by pipetting up and down several times with 5 ml pipet with pipette pump.
    6. Spin down at 520 x g for 5 min, then at 300 x g for 3 min to pellet the heterochronic cell mixtures (Figure 1F).

  4. Culturing (Video 4)
    1. Aliquot 1.5 ml REM per well of a 24-well plate.
    2. Cut the tip of the extra long transfer pipet with scissors, then flame the cut site to smoothen the surface. This will prevent damaging the pellet, and cells adhering to the pipet surface.
    3. After spinning down the heterochronic cell aggregate (from step C6), dislodge the aggregate from the bottom of the tube by taking some liquid off with a P1000 pipetman. Gently dispense liquid back into the tube at the edge of the pellet in order to dislodge the pellet (see Notes 6 and 7).
    4. Place dry Nucleopore Track-Etch Membrane on the sterilized Kimwipe sitting on sterile Petri dish lid. Place the tip of the Dumont forceps AA on the edge of the membrane to prevent membrane from rolling when cell pellet is transferred.
    5. Transfer cell pellet with extra long transfer pipet onto the centre of a dry Nuclepore Track-Etch Membrane (Figure 1G). Note that it is extremely critical to drop the pellet on the centre of the membrane. If pellet is dropped on the edge of the membrane, you could lose the cell pellet as it will flow towards Kimwipe and away from the membrane. Kimwipe should absorb the liquid leaving only the pellet on the membrane.
    6. Dry the membrane further by placing the membrane on a sterilized Kimwipe. Leave no trace of liquid on the membrane, as it will increase the chance of the membrane sinking in the media, which will result in cell death.
    7. Grab edge of the membrane with the cell pellet using Dumont forceps AA and gently place it onto the surface of the REM in one well of the 24-well plate (Figure 1H). Make sure the membrane is floating on the media, and does not sink as cells in the pellet will die if submersed.
    8. Incubate cell aggregate at 37 °C, 5% CO2 for 8 days in vitro (DIV). Replace ½ of the media (750 µl) every second day. Note that it is critical that the membrane is never submerged, so extra care is required when transferring plates in and out of the incubator.

      Video 4. Transferring heterochronic cell aggregate onto the membrane

  5. Cell collection and analysis
    1.  Replace REM with 1 ml of 1x DPBS to wash aggregate. Rinse with 1x DPBS two more times to remove trace of REM. Make sure cell pellets are not dislodged from the membrane.
    2. Remove as much 1x DPBS as possible, then add 500 µl of 4% PFA. Fix aggregate overnight at 4 °C. Note that a shorter fixation time may be required for certain antibodies.
    3. Wash aggregates three times for 10 min with 1x PBS, then immerse into 20% sucrose/1x PBS for cryoprotection overnight at 4 °C.
    4. Embed aggregates on membranes into optical cutting temperature compound (OCT) and freeze on dry ice (Figure 3). The membrane should be embedded perpendicular to the bottom of the mold to give a radial section through the retina. We recommend embedding replicate aggregates into the same block.
    5. Section aggregate on a cryostat at 10 µm, and collect sections on Superfrost Plus slides. We usually collect approximately seven sections per slide on up to 50 slides per block. Place the slides into a Standard microscope slide box. Slides can either be immunostained directly or stored. For storage, tape slide box with masking tape to close completely and prevent moisture from entering. Store box in -20 °C freezer.


      Figure 3. Orientation of heterochronic cell aggregate on the membrane and in the mold when embedding into OCT

  6. Immunostaining
    1. Bring the slide box to room temperature for 30 min before opening, then remove slides.
    2. Place slides in pre-warmed 2 N hydrochloric acid and incubate at 37 °C for 20 min.
    3. Rinse slides in distilled H2O several times to remove as much hydrochloric acid as possible.
    4. Wash three times for 5 min in 1x PBS to equilibrate the pH of the slides.
    5. Remove slides from 1x PBS and apply paper towel on the edge of the slide to remove excess liquid.
    6. Apply 400 μl of blocking solution (10% horse serum/1x PBT) per slide and incubate at room temperature for 1 h. Make sure blocking solution is spread thoroughly to cover the entire surface of the slides.
    7. Remove as much blocking solution as possible, then apply 200 μl of primary antibodies diluted in blocking solution (see Table 1 for dilution) and place Parafilm to cover entire surface of the slides. Incubate at 4 °C overnight.
    8. Wash slides three times for 10 min in 1x PBS.
    9. Apply 200 μl of secondary antibodies (see Table 1) and DAPI diluted in 1x PBT (1:500 and 1:500 dilution respectively) and place Parafilm on top to cover solution. Incubate at room temperature for 1 h. Avoid incubating under light as fluorescence is light-sensitive.
    10. Wash slides three times for 10 min in 1x PBS.
    11. Apply Aqua-Poly/Mount and mount with coverslip.
    12. Take several images (3 or more) of sections of the retinal cell aggregates with upright fluorescence microscope (Figure 1I).

Data analysis

  1. For experimental design, to compare two data sets, we perform the pellet assay a minimum of three times. To compare three or more data sets, we perform a minimum of four experiments. For each experiment, we use a minimum of three replicates. For each replicate, we analyse 3-10 photomicrographs so as to count a minimum of 500 BrdU+ cells.
  2. For data analysis, to assess the fate of BrdU-labeled retinal progenitor cells, we analyse a minimum of 3 cryosections from each pellet (Figures 4A and 4B). We take photomicrographs of each immunostained cryosection, and count the total number of BrdU+ cells and the number of BrdU+ cells that express a cell type-specific marker. For amacrine cells, as an example, we use Pax6 as a cell type-specific marker (Figure 4A). The percentage of BrdU+ progenitor cells that differentiate into Pax6+ amacrine cells is calculated by the formula: %Pax6+BrdU+/BrdU+ cells (as in Tachibana et al., 2016).
  3. For statistical analysis, we use GraphPad Prism. To compare two data sets, we perform a Student’s t-test. To compare three data sets, we perform a One-way ANOVA and post-hoc Tukey correction (as in Tachibana et al., 2016).


    Figure 4. Data processing and analysis of Pax6+BrdU+ cells in the pellet. A. High magnification image of a cryosection of a pellet co-immunostained for Pax6 and BrdU. Arrow/Arrowheads point to BrdU+ cell (arrow; left), Pax6+ cell (white arrowhead; middle) and Pax6+BrdU+ cell (blue arrowhead; right). B. Representative image of how we process data. Number of BrdU+ and Pax6+BrdU+ cells are counted from multiple sections (at least three sections) per pellet, which are at least in triplicate. We combine the data from all sections from each replicate, and consider this as N = 1. To avoid bias, we repeat the experiment at least three times from different litters from different parents.

Notes

  1. CD1 male and female mice are crossed and embryos are staged considering the morning a vaginal plug is detected as E0.5. E15.5 CD1 embryos, for example, are collected 15 days after
    the day of the plug.
  2. Ptenfl/fl; Pax6-Cre+ or Pten+/+ mice are generated by crossing a Ptenfl/+ female with a Ptenfl/+; Pax6-Cre+ male, considering the day the pups are born as P0. P2 embryos are collected 2 days after the day of birth.
  3. No alterations to the protocol are required if different mouse strains are used.
  4. When removing RPE and lens from the eyes, it is extremely crucial not to damage the retina as you could lose some cells. While the eye is larger at P2 compared to E15.5, at the macroscopic level there are no visible differences between E15.5 and P2 eyes except size. Thus, the procedure to remove the RPE and lens from the retina remains the same.
  5. While the retina is easily dissociated with trypsin, other cell types might not be as easily dissociated. In this case, other agents may be tested for dissociation (e.g., collagenase, papain, TrypLETM). Cell strainers can be used to remove clumps as an alternative for tissues that are difficult to dissociate.
  6. When making the aggregate pellets, we highly recommend to make at least five replicates per sample as you may lose pellets during culturing (e.g., If they sink). This will allow you to have at least three replication controls for each experiment, which should provide a good indication of experimental variability.
  7. When dislodging the pellet from the bottom of the 15 ml tube, do not apply too much force as it is easy to break apart the pellet. We usually pipet 500 μl of liquid using a P1000 pipetman and slowly dispense liquid on the edge of the pellet. You should see the pellet float in the liquid and then it is easy to pick up.

Recipes

  1. 2.5% trypsin (40 ml)
    1. Weigh 1 g of trypsin powder in 50 ml tube and add 40 ml of 1x DPBS
    2. Vortex until the solution is fully dissolved
    3. Filter sterilize (0.22 μm), aliquot and store at -80 °C
    4. Dilute to 0.125% with 1x DPBS for working solution (make fresh prior to use)
  2. 20% FBS/1x DPBS (10 ml) (make fresh prior to use)
    In 8 ml 1x DPBS, add 2 ml FBS
    Filter sterilize (0.22 μm) and store at room temperature
  3. Retinal explant media (REM) (50 ml)
    Add all of the components:
    25 ml DMEM
    12.5 ml HBSS (1x)
    12.5 ml heat inactivated horse serum
    50 μl L-glutamine (200 mM)
    300 μl HEPES (1 M)
    500 μl penicillin-streptomycin (100x)
    100 μl amphotericin B
    Filter sterilize (0.22 μm) and store at 4 °C for up to 1 month
  4. 1 mg/ml BrdU (10 ml)
    1. Weigh 10 mg of BrdU in 15 ml tube and add 10 ml of 1x DPBS
    2. Vortex until the powder is fully dissolved
    3. Aliquot and store at -20 °C
  5. 10x phosphate-buffered saline (PBS) (2 L)
    1. Mix 163.6 g NaCl, 23 g Na2HPO4, 3.7 g KCl, 4.06 g KH2PO4
    2. Bring to 2 L with distilled H2O
    3. Autoclave for 20 min at 121 °C
    4. 10x PBS is kept at room temperature up to 1 year
    5. Dilute to 1x with distilled H2O, and adjust pH to 7.5 for working solution
    6. The 1x PBS can be kept at room temperature for up to 3 months
  6. 20% paraformaldehyde (PFA) (200 ml)
    1. Weigh 40 g of crystalline PFA in a beaker and add 200 ml of 1x PBS
    2. Heat the solution until the powder is fully dissolved to 65 °C (not higher as PFA will degrade)
    3. Aliquot and store at -20 °C
    4. Dilute to 4% with 1x PBS and adjust pH to 7.5 for working solution (make fresh prior to use)
  7. 20% sucrose (500 ml)
    1. Weigh 100 g of sucrose in a beaker and add 500 ml of 1xPBS
    2. Heat the solution and mix gently until the powder is fully dissolved
    3. Filter sterilize (Bottle Top) and store at 4 °C up to 6 months
  8. 2 N hydrochloric acid (118 ml) (make fresh)
    1. Measure 98 ml distilled H2O in a flask and add 20 ml hydrochloric acid gently in the fume hood
    2. Pre-warm to 37 °C prior to BrdU immunostaining
  9. 1x phosphate-buffered saline/0.1% Triton X-100 (PBT) (2 L)
    1. Dilute 10x PBS (200 ml) to 1x with distilled H2O (1,800 ml)
    2. Add 2 ml of Triton X-100 and adjust pH to 7.5
    3. The solution can be kept at room temperature for up to 2 weeks
  10. Blocking solution (10% horse serum/1x PBT) (5 ml) (make fresh prior to use)
    Aliquot 500 μl of horse serum into 15 ml tube and add 4.5 ml 1x PBT
    Vortex to mix thoroughly
  11. Primary antibodies are diluted in blocking solution as described in Table 1
  12. Secondary antibodies are diluted 1/500 in PBT. DAPI pre-made commercial solution is added at 1/500. Secondary antibodies are listed in Table 1.

    Table 1. List of primary and secondary antibodies

Acknowledgments

This work was supported by grants from Brain Canada to CS and VW and the Canadian Institute of Health Research (CIHR) (Grant #89994) and Lion’s Sight Centre to CS. CS is the Dixon Family Chair in Ophthalmology Research at Sunnybrook Research Institute. NT was supported by an Alberta Children’s Hospital Research Institute (ACHRI)-CIHR scholarship. This protocol was adapted from procedures published in Tachibana et al. (2016) and Ma et al. (2007).

References

  1. Belliveau, M. J. and Cepko, C. L. (1999). Extrinsic and intrinsic factors control the genesis of amacrine and cone cells in the rat retina. Development 126(3): 555-566.
  2. Harmon, E. B., Apelqvist, A. A., Smart, N. G., Gu, X., Osborne, D. H. and Kim, S. K. (2004). GDF11 modulates NGN3+ islet progenitor cell number and promotes beta-cell differentiation in pancreas development. Development 131(24): 6163-6174.
  3. Jensen, A. M. and Wallace, V. A. (1997). Expression of Sonic hedgehog and its putative role as a precursor cell mitogen in the developing mouse retina. Development 124(2): 363-371.
  4. Lui, J. C. and Baron, J. (2011). Mechanisms limiting body growth in mammals. Endocr Rev 32(3): 422-440.
  5. Ma, L., Cantrup, R., Varrault, A., Colak, D., Klenin, N., Gotz, M., McFarlane, S., Journot, L. and Schuurmans, C. (2007). Zac1 functions through TGFbetaII to negatively regulate cell number in the developing retina. Neural Dev 2: 11.
  6. Reh, T. A. and Tully, T. (1986). Regulation of tyrosine hydroxylase-containing amacrine cell number in larval frog retina. Dev Biol 114(2): 463-469.
  7. Ringuette, R., Wang, Y., Atkins, M., Mears, A. J., Yan, K. and Wallace, V. A. (2014). Combinatorial hedgehog and mitogen signaling promotes the in vitro expansion but not retinal differentiation potential of retinal progenitor cells. Invest Ophthalmol Vis Sci 55(1): 43-54.
  8. Tachibana, N., Cantrup, R., Dixit, R., Touahri, Y., Kaushik, G., Zinyk, D., Daftarian, N., Biernaskie, J., McFarlane, S. and Schuurmans, C. (2016). Pten regulates retinal amacrine cell number by modulating Akt, Tgfbeta, and Erk signaling. J Neurosci 36(36): 9454-9471.
  9. Tobin, J. F. and Celeste, A. J. (2005). Myostatin, a negative regulator of muscle mass: implications for muscle degenerative diseases. Curr Opin Pharmacol 5(3): 328-332.
  10. Wallace, V. A. (2011). Concise review: making a retina--from the building blocks to clinical applications. Stem Cells 29(3): 412-417.
  11. Waid, D. K. and McLoon, S. C. (1998). Ganglion cells influence the fate of dividing retinal cells in culture. Development 125(6): 1059-1066.
  12. Wang, Y., Dakubo, G. D., Thurig, S., Mazerolle, C. J. and Wallace, V. A. (2005). Retinal ganglion cell-derived sonic hedgehog locally controls proliferation and the timing of RGC development in the embryonic mouse retina. Development 132(22): 5103-5113.
  13. Watanabe, T. and Raff, M. C. (1990). Rod photoreceptor development in vitro: intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina. Neuron 4(3): 461-467.
  14. Watanabe, T., Voyvodic, J. T., Chan-Ling, T., Sagara, H., Hirosawa, K., Mio, Y., Matsushima, S., Uchimura, H., Nakahara, K. and Raff, M. C. (1997). Differentiation and morphogenesis in pellet cultures of developing rat retinal cells. J Comp Neurol 377(3): 341-350.
  15. Wu, H. H., Ivkovic, S., Murray, R. C., Jaramillo, S., Lyons, K. M., Johnson, J. E. and Calof, A. L. (2003). Autoregulation of neurogenesis by GDF11. Neuron 37(2): 197-207.
  16. Zhang, X. M. and Yang, X. J. (2001). Regulation of retinal ganglion cell production by Sonic hedgehog. Development 128(6): 943-957.

简介

构成成熟视网膜的所有七种视网膜细胞类型都是由普通的多能视网膜祖细胞池(RPC)产生的(Wallace,2011)。已经产生足够数量的特定细胞类型的RPC知道的一种方式是通过负反馈信号,其由分化细胞发射并且必须达到阈值水平以阻止该细胞类型的额外分化。评估负反馈信号是否由分化细胞发出的关键测定是异源沉淀测定,其中早期RPC被解离并用BrdU标记,然后与20倍过量的解离的分化细胞混合。然后将组合的细胞再次聚集并在细胞膜上培养7-10天。在这段时间内,RPC将会分化,BrdU + RPC的命运可以使用细胞类型特异性标记进行评估。开发这种沉淀测定的研究人员最初表明,当两种细胞类型混合在一起时,新生儿RPC与胚胎RPC相比,在加速进度条件下产生杆(Watanabe和Raff,1990; Watanabe等,1997)。我们已经使用这种测定来证明我们发现作为视网膜神经节细胞(RGC)分化的负调节物的声刺猬(Shh)促进RPC增殖(Jensen和Wallace,1997; Ringuette等,2014)。最近我们修改了异质性沉淀测定法,以评估视网膜无长突细胞的反馈信号的作用,将转化生长因子β2(Tgfβ2)鉴定为负反馈信号,并将Pten作为Tgfβ2应答的调节剂(Ma et al。,2007 ; Tachibana等,2016)。该测定可以适应其他谱系和组织以评估两种不同细胞类型(异型)之间的细胞 - 细胞间的相互作用,以等时或异时的方式。
【背景】使用几种机制来确保在器官和组织发育过程中产生正确数量的分化细胞。例如,祖细胞可以响应分化细胞分泌的激素或生长因子的水平,祖细胞可以计数其经历的分裂数,或者可能存在计数分化细胞的最终数量的机制(Lui和Baron,2011 )。在视网膜中,由分化细胞分泌的负反馈信号由祖细胞感测,当信号达到阈值水平时,祖细胞停止产生分化细胞类型(Belliveau和Cepko,1999; Reh和Tully,1986; Waid和McLoon,1998 )。我们等已经证明,Shh是RGC命运的重要负面调节器(Wang et al。,2005; Zhang and Yang,2001)。我们还解剖了视网膜无长突细胞的反馈过程,表明转录因子Zac1作用于无长突细胞,以启动转化生长因子b2(Tgfb2)表达,其负调节RPC增殖和无长突细胞分化(Ma et al。,2007)。值得注意的是,其他TGFβ家族成员在嗅觉上皮(Wu et al。,2003),胰腺(Harmon et al。,2004)和骨骼肌(Tobin和Celeste,2005)中具有相似的反馈功能。我们还使用异质沉淀测定法来检查无畸变细胞反馈信号如何被调节。我们发现Pten是无长突细胞分化的重要正调节因子,并使用沉淀测定,我们证明Pten作用于RPC以控制对Tgfβ2信号传导的反应(Tachibana等,2016)。了解无角蛋白细胞和RPC相互作用如何提供对视网膜中细胞数量如何控制的重要新见解。值得注意的是,Pten和Tgfβ信号传导之间的类似相互作用可能是其他脊椎动物器官中细胞数量控制的基础,其中Tgfβ信号传导是器官大小的重要决定因素。

关键字:异时性细胞团分析法, 视网膜分化, 视网膜祖细胞, 重新聚合, 无长突细胞, 负反馈信号, 异型细胞相互作用

材料和试剂

  1. Fisherbrand无菌100 x 15 mm聚苯乙烯培养皿(Thermo Fisher Scientific,Fisher Scientific,目录号:FB0875713)
  2. 15ml锥形管(SARSTEDT,目录号:62.554.502)
  3. 无菌单独包装5ml移液器(SARSTEDT,目录号:86.1253.001)
  4. 组织培养24孔板(SARSTEDT,目录号:83.3922.300)
  5. Samco TM超长转移移液管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:262)
  6. 13 mm,0.8μmNucleopore Track-Etch膜(GE Healthcare,目录号:110409)
  7. Kimwipes(Kimberly-Clark Worldwide,目录号:34120)(不经高压灭菌,但保持清洁)
  8. Superfrost Plus Micro幻灯片(VWR,目录号:48311-703)
  9. 标准显微镜幻灯片盒(希思罗科学,目录号:HEA15991A)
  10. 微盖玻璃(VWR,目录号:48404-454)
  11. 4 in x 250 ft Parafilm roll(Bemis,目录号:PM999或VWR,目录号:52858-032)
  12. 50毫升康宁管(SARSTEDT,目录号:62.547.254)
  13. 0.22μm灭菌过滤器过滤(SARSTEDT,目录号:83.1826.001)
  14. 60 ml注射器(Medtronic,目录号:8881560125)
  15. 快速流动 TM无菌一次性瓶顶过滤器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:291-4520)
  16. E15.5 CD1小鼠(Charles River Laboratories International,目录号:022)(见注1)
  17. p ten ten ten;;;;;;;;;;;;;;; pax6-Cre + Pten +/+ 鼠标(见注2)
  18. 1×Ca 2+ +/sup>/Mg 2+无自由DPBS(Thermo Fisher Scientific,Gibco TM,目录号:14190250)
  19. 台盼蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
  20. Aquapolymount(Polysciences,目录号:18606)
  21. 胰蛋白酶(Sigma-Aldrich,目录号:T1005)
  22. 热灭活的FBS(胎牛血清)(Thermo Fisher Scientific,Gibco TM,目录号:12484028)
  23. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11965092)
  24. 1x HBSS(Thermo Fisher Scientific,Gibco TM ,目录号:24020117)
  25. 热灭活马血清(Thermo Fisher Scientific,Gibco TM,目录号:26050088)
  26. 200mM L-谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  27. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  28. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  29. 两性霉素B(Thermo Fisher Scientific,Gibco TM ,目录号:15290026)
  30. 5-溴嘌呤(BrdU)(Sigma-Aldrich,目录号:850187)
  31. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  32. 氯化钾(KCl)(EMD Millipore,目录号:PX1405)
  33. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5379)
  34. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
  35. 结晶PFA(Sigma-Aldrich,目录号:P6148)
  36. 蔗糖(Sigma-Aldrich,目录号:S9378)
  37. 11.8M盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  38. Triton X-100(Sigma-Aldrich,目录号:T8787)
  39. 4',6-二脒基-2-苯基吲哚,二盐酸盐(DAPI)作为如制造商(Thermo Fisher Scientific,Molecular Probes ,目录号:D1306)所述使用的预制的工作溶液,
  40. 抗Pax6和BrdU的抗体(见配方,表1)
    Pax6(BioLegend,目录号:901301)
    BrdU(Bio-Rad Laboratories,目录号:OBT0030)
    驴抗兔Alexa-Fluor 488(Thermo Fisher Scientific,Invitrogen TM,目录号:A-21206)
    山羊抗大鼠Alexa Fluor 568(Thermo Fisher Scientific,Invitrogen TM,目录号:A-11077)
  41. 最佳切割温度复合(VWR,目录号:95057-838)
  42. 2.5%胰蛋白酶(见食谱)
  43. 0.125%胰蛋白酶(见配方)
  44. 视网膜外植体介质(REM)(见配方)
  45. 1 mg/ml BrdU(见配方)
  46. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  47. 1x磷酸缓冲盐水(PBS)(见配方)
  48. 20%多聚甲醛(PFA)(见配方)
  49. 4%多聚甲醛(PFA)(见配方)
  50. 20%蔗糖(见食谱)
  51. 2 N盐酸(见食谱)
  52. 1x磷酸盐缓冲盐水/0.1%Triton X-100(PBT)(参见食谱)
  53. 阻塞解决方案(见配方)

设备

  1. Dumont镊子#5(精细科学工具,目录号:11252-20)
  2. 杜蒙镊子#55(精细科学工具,目录号:11255-20)
  3. Dumont镊子AA(精细科学工具,目录号:11210-20)
  4. 浅水摇晃水浴(Precision Scientific,目录号:66799)
  5. 移液泵(SP科学软件 - 贝尔艺术产品 - H-B仪器,目录号:F37898-0000)
  6. 37℃,5%CO 2水夹套培养箱(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3110)
  7. 15ml角管的冷藏台式离心机(Eppendorf,型号:5810 R)
  8. 血细胞计数室(Hausser Scientific,目录号:3100)
  9. P20移液器(Gilson,目录号:F123600)
  10. P200移液器(Gilson,目录号:F123601)
  11. P1000移液器(Gilson,目录号:F123602)
  12. 冷冻机(Leica Biosystems,型号:CM3050 S)
  13. -20°C冷冻机
  14. 直立荧光显微镜(Leica Microsystems,型号:DM RXA2)
  15. 高压灭菌器
  16. 1升烧杯(Corning,Pyrex ®,目录号:1395-1L)
  17. 500ml烧杯(Corning,Pyrex ®,目录号:1395-500)
  18. 250ml锥形烧瓶(Corning,Pyrex ,目录号:4450-250)
  19. 通风柜
  20. 立体显微镜用于解剖(Leica Microsystems,型号:MZ6)
  21. 倒置光学显微镜(Leica Microsystems,型号:DMIL LED)

软件

  1. GraphPad Prism(GraphPad软件)

程序

注意:

  1. 在实验前,预热0.125%胰蛋白酶和REM(见配方)至37°C。
  2. 这种方案是在没有无菌技术的情况下进行的,但我们非常小心,以尽可能地保持细胞和设备的清洁。
  1. 分离E15.5小鼠视网膜
    1. 在室温从E15.5 CD1胚胎中取出眼睛(见注1关于小鼠)
    2. 将眼睛放在培养皿中,然后加上1×DPBS以覆盖(图1A)
    3. 使用Dumont镊子#55和#5(图1B)从眼睛中去除视网膜色素上皮(RPE)和晶状体(见附注4)。要删除RPE,用Dumont镊子#55抓住一小块巩膜,然后用Dumont镊子#5刺入巩膜以引起切口。用镊子研磨切口的边缘,并缓慢撕开切口。继续打开切口,直到去除巩膜,角膜和视神经。 RPE应与角膜一起脱离视网膜(视频1)。保持眼睛冷1×DPBS帮助RPE脱离。使用镊子从视网膜上取下睫毛边缘。这会松开视网膜上的镜片附件。轻轻拉出镜头(视频2)。为每个眼睛解剖替换1x DPBS,以保持培养皿和缓冲液冷。

      <! - flashid2117v162开始 - >
      视频1.从眼睛中删除RPE
      <! - [if!IE]> - > <! - <![endif] - >

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      Get Adobe Flash Player

      - ! - [if!IE]> - >
      <! - <![endif] - >
      <! - flashid2117v162结束 - >
      <! - flashid2117v163开始 - >
      视频2.从眼睛中取出镜头
      <! - [if! > - > <! - <![endif] - >

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      Get Adobe Flash Player

      - ! - [if!IE]> - >
      <! - <![endif] - >
      <! - flashid2117v163结束 - >
    4. 将视网膜转移到15ml康宁管中(室温下用10ml的DPBS)(图1C)。让视网膜沉入管底部。 (我们建议收集〜20个视网膜[10个胚胎的2只眼睛],这样可以获得约8.0×10 6个细胞。)
    5. 使用5ml移液器移除尽可能多的1x DPBS。确保不要移出视网膜组织。
    6. 向视网膜中加入100μl0.125%胰蛋白酶(预温至37℃),并在37℃水浴中孵育10分钟(图1D)。请注意,视网膜本身不会分开。破碎细胞间相互作用是必需的。
    7. 加入5毫升20%FBS/1×DPBS以停止胰蛋白酶消化
    8. 通过使用5ml移液管(图1E),使用5ml移液管(图1E)将上下移动数次(,至少30次)进行均质化,使视网膜细胞完全均匀宏观观察细胞簇。确保轻轻地研磨以避免任何气泡,因为气泡会破坏细胞。我们建议使用移液泵来确保研磨温和。 (视频3)(见注5)

      <! - flashid2117v164开始 - >
      视频3.研究胰蛋白酶消化的视网膜细胞
      <! - [if!IE]> - > <! - <![endif] - >

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      Get Adobe Flash Player

      - ! - [if!IE]> - >
      <! - <![endif] - >
      <! - flashid2117v164结束 - >
    9. 以520 x g旋转细胞5分钟,然后尽可能多地除去溶液。
    10. 将细胞重新悬浮在1ml REM(预热至37℃)中
    11. 将3μl1 mg/ml BrdU加入细胞再悬浮液中。涡旋温和地短暂混合。
    12. 在水夹套培养箱中,37℃,5%CO 2孵育细胞1小时。

  2. 分离P2鼠视网膜
    1. 同时,通过摘除眼睛解剖P2小鼠视网膜,并如上所述去除RPE和晶状体(参见关于小鼠的注2和注3)。
    2. 将视网膜转移到15ml康宁管中(室温下用10ml的DPBS),每只小鼠的基因型不同,将每只小鼠的两个视网膜保持在单独的管中。允许视网膜下沉到管底部。
    3. 去除尽可能多的1×DPBS,然后加入100μl的0.125%胰蛋白酶,并在37℃水浴中孵育10分钟。
    4. 通过加入5ml 20%FBS/1XDPBS停止反应,并用5ml移液管和移液管轻轻移液数次培养细胞。
    5. 以520 x x 旋转5分钟,尽可能多地移除解决方案。
    6. 用1ml REM重新悬浮细胞(预热至37℃)
  3. 异位细胞沉淀的制备
    1. 在BrdU孵育1小时后,从培养箱中取出E15.5 CD1解离细胞
    2. 以520×g旋转细胞5分钟,尽可能多地除去培养基,然后在1ml REM中重新悬浮。重复此步骤两次以完全删除任何BrdU痕迹。将细胞重新悬浮在1ml REM(预热至37℃)中
    3. 对于E15.5和P2细胞,取10μl细胞再悬浮并与10μl台盼蓝混合。使用血细胞计数器计算每个细胞再悬浮液的浓度(计算如图2所示)。确保细胞完全解离(即,,没有细胞团或双峰)。如果你看到任何一个块,我们建议重新解离单元格。
    4. 在REM中稀释E15.5细胞重悬浮至1×10 6细胞/ml。将1×10 6细胞(1ml)分批加入15ml Corning管中作为对照。将E15.5细胞再悬浮稀释至0.5×10 6细胞/ml。
    5. 在REM中稀释P2细胞重悬浮至1.0×10 6细胞/ml。将等分试样1×10 6细胞(1ml)加入15ml康宁管中。从E15.5 CD1细胞再悬浮液中加入0.5μl10μg/ml细胞的100μl(5.0×10 4个细胞)。用移液器泵用5ml移液管上下移动数次,彻底混匀。
    6. 以520×g旋转5分钟,然后在300×g下旋转3分钟以沉淀异源细胞混合物(图1F)。

  4. 培养(视频4)
    1. 24孔平板每孔1.5毫升REM。
    2. 用剪刀切割超长移液管的尖端,然后点燃切割部位以使表面光滑。这样可以防止颗粒破坏,细胞粘附在移液管表面
    3. 在旋转异源细胞聚集体(来自步骤C6)之后,通过用P1000移液管取出一些液体来从管的底部排出骨料。轻轻地将液体重新分配到颗粒边缘的管中,以便去除颗粒(参见注释6和7)。
    4. 将干燥的Nucleopore Track-Etch膜置于无菌培养皿盖上的灭菌Kimwipe上。将杜蒙镊子AA的尖端放在膜的边缘,以防止细胞沉积物转移时膜的滚动。
    5. 将转移细胞沉淀物用超长的转移移液管移至干燥的Nuclepore Track-Etch膜的中心(图1G)。请注意,将颗粒放在膜的中心是非常关键的。如果颗粒滴在膜的边缘,您可能会丢失细胞团块,因为它将流向Kimwipe并远离膜。 Kimwipe应吸收只留下薄膜上的颗粒的液体。
    6. 通过将膜放置在灭菌的Kimwipe上进一步干燥膜。不要在膜上留下液体痕迹,因为会增加介质中膜下沉的可能性,这将导致细胞死亡。
    7. 使用Dumont镊子AA将细胞沉淀物的边缘抓住,并轻轻地将其置于24孔板的一个孔中的REM表面上(图1H)。确保膜在介质上浮动,并且不会沉没,因为如果浸没,颗粒中的细胞会死亡。
    8. 在37℃,5%CO 2孵育细胞聚集体8天,在体外(DIV)。每隔一天更换1/2的介质(750μl)。请注意,将膜从不浸没是至关重要的,因此在将板转入和移出培养箱时需要格外小心。

      <! - flashid2117v165开始 - >
      视频4.将异源细胞聚集体转移到膜上
      <! - [if!IE]> - > <! - <![endif] - >

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      Get Adobe Flash Player

      - ! - [if!IE]> - >
      <! - <![endif] - >
      <! - flashid2117v165结束 - >
  5. 细胞收集和分析
    1. 将REM替换为1 ml 1x DPBS,以清洗聚集体。用1x DPBS冲洗两次以清除痕量的REM。确保细胞颗粒不会从膜上移出。
    2. 尽可能多地除去1个DPBS,然后加入500μl的4%PFA。在4°C固化骨料过夜。请注意,某些抗体可能需要较短的固定时间。
    3. 用1x PBS洗涤聚集体三次,持续10分钟,然后浸入20%蔗糖/1×PBS中4℃冷冻保护过夜。
    4. 将聚合物在膜上嵌入光学切割温度复合物(OCT)中并在干冰上冷冻(图3)。膜应垂直于模具的底部嵌入,以通过视网膜产生径向截面。我们建议将复制聚合嵌入到同一个块中。
    5. 在10微米的低温恒温器上部分聚集,并收集Superfrost Plus载玻片上的部分。我们通常每个幻灯片收集大约七个部分,每个块最多可以有50个幻灯片。将幻灯片放入标准显微镜幻灯片盒中。幻灯片可以直接免疫染色或储存。对于存放,带有胶带的胶带滑动盒完全关闭并防止水分进入。储存于-20°C冰箱。

  6. 免疫染色
    1. 打开前,将滑块放入室温30分钟,然后取出幻灯片。
    2. 将载玻片置于预热的2N盐酸中,37℃下孵育20分钟
    3. 在蒸馏的H 2 O 2中漂洗载玻片几次以除去尽可能多的盐酸。
    4. 在1x PBS中洗涤3次5分钟,以平衡载玻片的pH
    5. 从1x PBS中取出载玻片,并在纸张边缘上涂抹纸巾,以清除多余的液体。
    6. 每个载玻片应用400μl封闭液(10%马血清/1×PBT),室温孵育1 h。确保阻塞解决方案彻底传播,以覆盖幻灯片的整个表面。
    7. 去除尽可能多的封闭溶液,然后应用200μl在封闭溶液中稀释的一抗(参见表1进行稀释),并将Parafilm覆盖载玻片的整个表面。在4℃下孵育过夜。
    8. 在1x PBS中洗三次,10分钟。
    9. 应用200μl二抗(见表1)和稀释于1×PBT(分别为1:500和1:500稀释)中的DAPI,并将Parafilm放在顶部以覆盖溶液。在室温下孵育1小时。避免在光照下孵化,因为荧光是敏感的
    10. 在1x PBS中洗三次,10分钟。
    11. 应用水上安装和安装与盖玻片。
    12. 用直立荧光显微镜拍摄视网膜细胞聚集体的几个图像(3个或更多)(图1I)。

数据分析

  1. 对于实验设计,为了比较两个数据集,我们进行沉淀测定至少三次。为了比较三个或更多的数据集,我们至少执行四个实验。对于每个实验,我们至少使用三次重复。对于每个重复,我们分析3-10个显微照片,以便计数至少500 BrdU + 细胞。
  2. 为了进行数据分析,为了评估BrdU标记的视网膜祖细胞的命运,我们从每个颗粒中分析了最少3次冷冻切片(图4A和4B)。我们拍摄每个免疫染色冷冻切片的显微照片,并计数表达细胞类型特异性标记物的BrdU + 细胞总数和BrdU + 细胞数。对于无长角细胞,作为示例,我们使用Pax6作为细胞类型特异性标记(图4A)。分化为Pax6 + 无长角细胞的BrdU + 祖细胞的百分比由下式计算:%Pax6 + BrdU /sup>/BrdU + 单元格(如Tachibana 等等,2016)。
  3. 对于统计分析,我们使用GraphPad Prism。要比较两个数据集,我们执行学生的测试。为了比较三个数据集,我们执行单因素方差分析和事后Tukey校正(如Tachibana ,2016)。


    图1.视网膜/视网膜细胞在方案不同时间点的代表性图像。 A. E15.5摘除后眼睛; B.E15.5视网膜去除RPE后; C. E15.5视网膜沉入15 ml康宁管底部; D.在37℃水浴中对视网膜进行胰蛋白酶处理10分钟;解离的视网膜细胞。 F.异位视网膜细胞聚集体沉淀在15毫升康宁管(箭头)的底部; G.放置在膜上的细胞沉淀(用红色虚线勾画); H.放置在REM表面上的膜上的细胞沉淀(侧视图);用BrdU(红色)和无长角细胞标记Pax6(绿色)标记的聚集体。 le:镜片,re:视网膜,RPE:视网膜色素上皮

    图2.使用血细胞计数器计算细胞再悬浮浓度的公式。注意,稀释因子为2,细胞再悬浮液用台盼蓝稀释(10μl+ 10μl)。


    图3.嵌入OCT后膜和模具中异质细胞聚集体的取向


    图4. Pax6的数据处理和分析 + BrdU + 细胞。 A.用于Pax6和BrdU的联合免疫染色的颗粒的冷冻切片的高倍放大图。箭头/箭头指向BrdU + 单元格(箭头;左),Pax6 + 单元格(白色箭头;中间)和Pax6 + BrdU + 单元格(蓝色箭头;右)。 B.我们如何处理数据的代表性形象。 BrdU + 和Pax6 + BrdU + 细胞从每个颗粒的多个部分(至少三个切片)计数,其至少在一式三份我们结合来自每个重复的所有部分的数据,并将其视为N = 1。为了避免偏见,我们从不同父母的不同细胞重复实验至少三次。

笔记

  1. CD1雄性和雌性小鼠交叉,胚胎分期考虑早晨,阴道塞被检测为E0.5。 E15.5 CD1胚胎,例如,在
    后15天收集 插头的一天。
  2. ten ten ten ten;;;;;;;;;;;;;;;;;; Pax6-Cre + Pten +/+ 鼠标是通过跨越Pten fl/+ 女用Pten fl/+ ; Pax6-Cre + 男性,考虑到小狗出生于P0的那一天。 P2胚胎在出生当天后2天收集。
  3. 如果使用不同的小鼠品系,则不需要对协议进行更改。
  4. 当从眼睛中取出RPE和镜片时,非常重要的是不会损伤视网膜,因为您可能会丢失一些细胞。虽然P2眼睛的眼睛大于E15.5,但在宏观水平上,E15.5和P2眼睛除大小外没有明显的差异。因此,从视网膜中去除RPE和镜片的步骤保持不变。
  5. 虽然视网膜容易与胰蛋白酶解离,但其他细胞类型可能不会很容易解离。在这种情况下,可以测试其它试剂的解离(例如,胶原酶,木瓜蛋白酶,TrypLE TM)。细胞过滤器可用于去除团块,作为难以解离的组织的替代品。
  6. 当制作聚集颗粒时,我们强烈建议每个样品至少进行五次重复,因为您可能会在培养过程中(例如,如果它们沉没)会损失颗粒。这将允许您对每个实验至少有三个复制控件,这应该提供实验变异性的良好指示
  7. 当从15毫升管的底部移除颗粒时,不要施加太大的力,因为它很容易分离颗粒。我们通常使用P1000移液管吸取500μl液体,并在颗粒边缘缓慢分配液体。你应该看到颗粒浮在液体中,然后很容易拿起。

食谱

  1. 2.5%胰蛋白酶(40ml)
    1. 称取1 ml胰蛋白酶粉末在50ml管中,并加入40 ml 1x DPBS
    2. 旋转直至溶液完全溶解
    3. 过滤消毒(0.22μm),等分并储存于-80°C
    4. 使用1x DPBS将溶液稀释至0.125%(使用前先清新)
  2. 20%FBS/1x DPBS(10ml)(使用前先清)
    在8ml 1x DPBS中,加入2ml FBS
    过滤灭菌(0.22μm),并在室温下保存
  3. 视网膜外植体培养基(REM)(50ml)
    添加所有组件:
    25 ml DMEM
    12.5 ml HBSS(1x)
    12.5 ml热灭活马血清
    50μlL-谷氨酰胺(200 mM)
    300μlHEPES(1 M)
    500μl青霉素 - 链霉素(100x)
    100μl两性霉素B
    过滤消毒(0.22μm),并在4°C下储存多达1个月
  4. 1mg/ml BrdU(10ml)
    1. 称取10毫克BrdU在15毫升管中,并加入10毫升1x DPBS
    2. 旋转直至粉末完全溶解
    3. 等分并储存于-20°C
  5. 10倍磷酸缓冲盐水(PBS)(2升)
    1. 混合163.6g NaCl,23g Na 2 HPO 4,3.7g KCl,4.06g KH 2 PO 4,
    2. 带蒸馏H 2 O 2/2的2升
    3. 在121°C高压灭菌20分钟
    4. 10倍PBS在室温下保存至1年
    5. 用蒸馏的H 2 O稀释至1倍,并将工作溶液的pH值调节至7.5
    6. 1x PBS可以在室温下保存3个月以上
  6. 20%多聚甲醛(PFA)(200ml)
    1. 在烧杯中称量40 g结晶PFA,加入200 ml 1x PBS
    2. 加热溶液直到粉末完全溶解至65°C(不高于PFA会降解)
    3. 等分并储存于-20°C
    4. 用1x PBS稀释至4%,并将工作溶液的pH调至7.5(使用前先清新)
  7. 20%蔗糖(500ml)
    1. 在烧杯中称量100g蔗糖,并加入500 ml 1xPBS
    2. 加热溶液并轻轻混合,直至粉末完全溶解
    3. 过滤消毒(瓶顶),并在4°C下储存6个月
  8. 2N盐酸(118ml)(使新鲜)
    1. 在烧瓶中测量98ml蒸馏的H 2 O 2,并在通风橱中轻轻加入20ml盐酸。
    2. 在BrdU免疫染色之前预热至37°C
  9. 1x磷酸缓冲盐水/0.1%Triton X-100(PBT)(2L)
    1. 用蒸馏的H 2 O(1,800ml)稀释10倍的PBS(200毫升)至1倍。
    2. 加入2ml Triton X-100并调节pH至7.5
    3. 该溶液可以在室温下保存长达2周
  10. 阻塞溶液(10%马血清/1×PBT)(5ml)(使用前使新鲜)
    将500μl马血清分成15ml管,加入4.5ml 1x PBT 涡旋混合彻底
  11. 将初级抗体以封闭溶液稀释,如表1中所述
  12. 二抗在PBT中稀释1/500。 DAPI预制商业解决方案以1/500添加。第二抗体列于表1中
    表1.主要和次要抗体列表

致谢

这项工作得到了加拿大脑力量大学和加拿大大学以及加拿大希思研究所(CIHR)(拨款号89994)和狮子会视觉中心的支持。 CS是Sunnybrook研究所眼科研究中的Dixon家庭主席。新罕布什尔州得到艾伯塔省儿童医院研究所(ACHRI)-CIHR奖学金的支持。该方案根据Tachibana等人发表的程序进行了改进。 (2016)和Ma等人(2007)。

参考文献

  1. Belliveau,MJ和Cepko,CL(1999)。外在的和内在因素控制大鼠视网膜中无角蛋白和锥细胞的发生。发展 126(3):555-566。
  2. Harmon,EB,Apelqvist,AA,Smart,NG,Gu,X.,Osborne,DH和Kim,SK(2004)。  GDF11调节NGN3 + 胰岛祖细胞数,促进胰腺发育中的β细胞分化。/em> 131(24):6163-6174。
  3. Jensen,AM和Wallace,VA(1997)。  表达式的声波刺猬及其在发展中的小鼠视网膜中作为前体细胞丝裂原的推定作用。发展 124(2):363-371。
  4. Lui,JC和Baron,J.(2011)。  限制哺乳动物体内生长的机制。 Endocr Rev 32(3):422-440。
  5. Ma,L.,Cantrup,R.,Varrault,A.,Colak,D.,Klenin,N.,Gotz,M.,McFarlane,S.,Journot,L。和Schuurmans,C.(2007)通过TGFbetaII Zac1 功能负调节发展中的视网膜中的细胞数目。神经元 2:11.
  6. Reh,TA和Tully,T。(1986)。调控幼虫视网膜中含酪氨酸羟化酶的无长突细胞数目。 Dev Dev。114(2):463-469。
  7. Ringuette,R.,Wang,Y.,Atkins,M.,Mears,AJ,Yan,K.and Wallace,VA(2014)。  55(1):43-54。
  8. Tachibana,N.,Cantrup,R.,Dixit,R.,Touahri,Y.,Kaushik,G.,Zinyk,D.,Daftarian,N.,Biernaskie,J.,McFarlane,S。和Schuurmans,C( 2016)。 Pten 规范视网膜无长突细胞数量通过调节Akt,Tgfbeta和Erk信号传导。 J Neurosci 36(36):9454-9471。
  9. Tobin,JF和Celeste,AJ(2005)。  肌生成抑制素,肌肉量的负调节物:对肌肉退行性疾病的影响。 Curr Opin Pharmacol 5(3):328-332。
  10. 华莱士,VA(2011)。简明回顾:制作视网膜 - 从构建块到临床应用。 干细胞 29(3):412-417。
  11. Waid,DK and McLoon,SC(1998)。  神经节细胞影响分化培养的视网膜细胞的命运。发展 125(6):1059-1066。
  12. Wang,Y.,Dakubo,GD,Thurig,S.,Mazerolle,CJ和Wallace,VA(2005)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih视网膜神经节细胞来源的声波刺猬在局部控制胚胎小鼠视网膜中RGC发育的增殖和时机。发展 132(22) ):5103-5113。
  13. Watanabe,T.和Raff,MC(1990)。棒状光感受器在体外发展:增殖神经上皮细胞的固有特性随着大鼠视网膜的发展而发生变化。 4(3):461-467。 br />
  14. Watanabe,T.,Voyvodic,JT,Chan-Ling,T.,Sagara,H.,Hirosawa,K.,Mio,Y.,Matsushima,S.,Uchimura,H.,Nakahara,K.and Raff,MC( 1997)。发育大鼠的颗粒培养物中的分化和形态发生视网膜细胞。 J Comp Neurol 377(3):341-350。
  15. Wu,HH,Ivkovic,S.,Murray,RC,Jaramillo,S.,Lyons,KM,Johnson,JE and Calof,AL(2003)。  GDF11的神经发生自动调节。神经元 37(2):197-207。 />
  16. Zhang,XM and Yang,XJ(2001)。  法规的视网膜神经节细胞生产由Sonic刺猬发展 128(6):943-957。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 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. Tachibana, N., Zinyk, D., Ringuette, R., Wallace, V. and Schuurmans, C. (2017). Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina. Bio-protocol 7(3): e2117. DOI: 10.21769/BioProtoc.2117.
  2. Tachibana, N., Cantrup, R., Dixit, R., Touahri, Y., Kaushik, G., Zinyk, D., Daftarian, N., Biernaskie, J., McFarlane, S. and Schuurmans, C. (2016). Pten regulates retinal amacrine cell number by modulating Akt, Tgfbeta, and Erk signaling. J Neurosci 36(36): 9454-9471.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。