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Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS
FACS法进行拟南芥精子和营养细胞核的样本制备和分离   

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Abstract

One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 106 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.

Keywords: SYBR Green(荧光染料掺入法), Fluorescence-activated cell sorting (FACS)(荧光激活细胞分选(FACS)), Arabidopsis(拟南芥), Pollen(花粉), Male gametophyte(雄配子体)

Materials and Reagents

  1. Plastic ware / consumables
    1. 15 ml polypropylene tubes (SARSTEDT AG & Co, catalog number: 62554502 )
    2. 50 ml polypropylene tubes (SARSTEDT AG & Co, catalog number: 62548004 )
    3. 100 µm nylon mesh (Biologix Group Limited, catalog number: 151100 )
    4. 40 µm nylon mesh (Biologix Group Limited, catalog number: 151040 )
    5. 250 ml centrifuge tubes (Bartelt, catalog number: 9.315 721 )
    6. 1.5 ml and 2 ml microliter reaction tubes
    7. Syringe filters 0.2 µm (Bartelt, catalog number: 9055511 )
    8. BD Falcon® 12 x 75 mm tube with cell strainer cap (BD Biosciences, catalog number: 352235 )
      Note: Currently, it is “Corning Inc., catalog number: 352235”.
    9. Sterican disposable hypodermic needles with a diameter of 0.45 x 25 mm (B. Braun Melsungen AG, Braun Petzold, catalog number: 4657683 )
    10. Pipettes tips (1,000 µl, 200 µl, 20 µl, 2 µl)
    11. Acid-washed glass beads (see Recipes)

  2. Glassware
    1. Glass beads 0.4-0.6 mm (Sartorius AG, catalog number: BBI8541701 )
    2. 250 ml Erlenmeyer flask
    3. Glass tubes
    4. Glass slides
    5. Cover slips

  3. Plant material
    1. 100 Flowering wild-type Arabidopsis thaliana plants (Col-0 accession and dml-3 mutant, are used as examples) (Arabidopsis, catalog number: SALK_056440 )

  4. Chemicals
    1. D(+)-Sucrose for analysis (AppliChem GmbH, catalog number: 131621.0914 )
    2. DAPI (4', 6-Diamidino-2-Phenylindole, dihydrochloride) (Life Technologies, InvitrogenTM, catalog number: D1306 )
      Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306”.
    3. SYBR Green I nucleic acid stain stock solution (Life Technologies, catalog number: S7567 )
      Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: S7567”.
    4. Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: 93482 )
    5. Complete, EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11873580001 )
    6. Calcium chloride dihydrate (AppliChem GmbH, catalog number: A3587 )
    7. Ethylenediamine tetraacetic acid disodium salt dihydrate (Carl Roth GmbH + Co., catalog number: 8043 )
    8. Ethylene glycol-bis (2-aminoethylether)-N, N, N, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
    9. Ficoll® PM 400 (Sigma-Aldrich, catalog number: F4375 )
    10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    11. Magnesium acetate tetrahydrate (Sigma-Aldrich, catalog number: M5661 )
    12. Sodium chloride (EMD Millipore Corporation, catalog number: 106404 )
    13. Potassium chloride (Sigma-Aldrich, catalog number: P3911 )
    14. D-Sorbitol (Sigma-Aldrich, catalog number: S6021 )
    15. Spermine [N, N'-Bis-(3-aminopropyl)-1, 4-diaminobutan] (AppliChem GmbH, catalog number: A0672.0005 )
    16. TritonTM X-100 (Sigma-Aldrich, catalog number: 234729 )
    17. Tris-base (AppliChem GmbH, catalog number: A1086 )
    18. Nitric acid 65% (Carl Roth GmbH + Co., catalog number: X898.1 )
    19. Hydrochloric acid fuming 37% (Carl Roth GmbH + Co., catalog number: 4625.1 )
    20. Sodium Hydroxide pellets (AppliChem GmbH, catalog number: A3910 )
    21. Ultrapure water (MilliQ) (conductivity > 18 MΩ-1 cm-1)

  5. Buffers
    1. BD FACSFlow Sheath Fluid (BD Bioscience, catalog number: 342003 )
    2. Sucrose solution (see Recipes)
    3. Buffer A (see Recipes)
    4. Buffer B (see Recipes)
    5. Protease inhibitor stock solution (see Recipes)
    6. DAPI stock solution (see Recipes)

Equipment

  1. Instruments
    1. Beckman Coulter Avanti J-26XP centrifuge with the JA-10 rotor
    2. Table-top centrifuge
    3. Orbital shaker (VWR International, catalog number: 89032102 )
    4. Drying oven (EHRET GmbH, catalog number: 3108 )
    5. Bead-beater (Retsch GmbH, model: MM301 )
    6. Fluorescence microscope with 20x, 40x, and 100x objectives and a DAPI filter
    7. Flow cytometer: FACS Aria I (BD Biosciences) with a 70 µm nozzle and 70 psi sheath pressure, and a 488 nm coherent sapphire solid state 13 mW laser for excitation and a 530/30 nm band-pass filter for detection of SYBR green I

  2. General lab equipment
    1. Beaker
    2. 250 ml Erlenmeyer flask
    3. Glass tubes
    4. Sharp-edged scissors
    5. Forceps
    6. Air displacement pipettes
    7. Ice container

Software

  1. Flow cytometric software (BD Biosciences, model: FACS Diva 6.1.2)

Procedure

  1. Release of A. thaliana pollen from inflorescences [as previously described (Honys and Twell, 2003; Johnson-Brousseau and McCormick, 2004; Schoft et al., 2009) with modifications]
    1. ~100 plants (A. thaliana) were grown for 5 weeks at 16 h light/8 h dark cycles at 22 °C. Developmental stage of the plants corresponds to 6.10 to 6.50 according to Table 2 in Boyers et al. (2001).
    2. All inflorescences were collected in a beaker (placed on ice) by cutting them off with sharp-edged scissors.
    3. 9% ice-cold sucrose solution was poured onto the collected inflorescences until they were covered. The inflorescences were vigorously swirled for one minute to release mature pollen grains.
    4. The suspension was filtered through a 100 µm nylon mesh into 250 ml centrifugation tubes placed on ice.
    5. Pollen grains were concentrated by centrifugation at 1,350 x g for 10 min at 4 °C.
    6. The supernatant was poured back onto the inflorescences, and steps 3-5 were repeated once more.
    7. The pollen pellets were resuspended in ice-cold buffer A to a final volume of 10 ml and filtered through a 40 µm nylon mesh into a 15 ml Falcon tube.
    8. Pollen grains were precipitated by centrifugation at 800 x g for 10 min at 4 °C.
    9. Pollen grains were washed once again with 1 ml of buffer A and collected in a sterile 1.5 ml tube by centrifugation at 5,000 x g for 5 min at 4 °C in a table-top centrifuge. The supernatant was discarded.
      Notes:
      1. Collecting inflorescences and releasing pollen takes about 2-3 h.
      2. The precipitated pollen can be used immediately or can be rapidly frozen in liquid nitrogen and kept at -80 ° C for several years.
      3. 100 A. thaliana plants (Col-0 ecotype) yield about 100 µl of pollen. Pollen pellet volume is estimated by filling an identical, empty tube with water up to the same level as the pollen and measuring the water volume with an air-displacement pipet. This amount can vary depending on accession, growth conditions, and age of the plant.

  2. Isolation of A. thaliana pollen nuclei from mature pollen
    1. 10-20 sterile 2 ml centrifuge tubes were each filled with 1.8 g of acid-washed glass beads. Unused tubes can be stored at room temperature and used later.
    2. Harvested pollen were mixed with buffer A supplemented with PMSF and protease inhibitors using the following pollen to buffer A ratios:
      Below a pollen pellet volume of 80 µl a ratio of 1:3 (pollen:buffer A) was used between 80 µl and 150 µl a ratio of 1 : 3.5 was used above 150 µl, a ratio of 1 : 4 was used for resuspension of pollen grains in buffer A.
    3. The pollen grain suspension was carefully pipetted into 2 ml centrifuge tubes filled with glass beads (prepared in step B1) in 50 µl aliquots.
    4. Pollen grains were subjected to bead beating by placing the 2 ml tubes filled with pollen suspension and glass beads in a Retsch ball mill and, subsequently, running a beating protocol for 1.5 min at 15 Hz.
    5.  Three holes were punched into the bottom of the tubes with a fresh needle. Lids were carefully cut off, and the 2 ml tubes were placed on top of sterile 1.5 ml tubes that have also had their lids removed. The two tubes were then placed into glass tubes for centrifugation, so that the nuclei suspension will be recovered in the 1.5 ml tube (alternatively, 15 ml Falcon tubes can be used) (Figure 1A).
    6. Glass tubes were centrifuged at 800 x g for 10 min at 4 °C. The 2 ml tubes containing the glass beads were discarded and the nuclei suspensions were pooled into a single sterile 1.5 ml centrifuge tube.
    7. 10 µl of pollen nuclei suspension was mixed with 1 µl of DAPI in water and pipetted onto a glass slide. A cover slip was carefully placed on top of the sample. Efficiency of pollen nuclei release and quality of nuclei were assessed with a fluorescence microscope using 20x and 40x objectives and the DAPI channel [excitation/emission of DAPI (nm): 358/461] (Figure 1B and C).


      Figure 1. Assessment of A. thaliana pollen nuclei release efficiency. A. Three-tube setup before and after centrifugation. B. Microscopic image of A. thaliana wild-type (Col-0) pollen before bead beating, DAPI: DAPI-stained pollen examined with the DAPI filter. 40x magnification. Size bar: 25 µm. C. Microscopic image of A. thaliana wild-type pollen (Col-0) after bead beating. Arrows: (1) intact pollen, (2) nuclei still attached to cytoplasm, (3) empty pollen shell, (4) released vegetative nucleus. DAPI: DAPI-stained pollen examined with the DAPI filter. 40x magnification. Size bar: 25 µm

      Notes:
      1. Centrifuge tubes should be kept at 4 °C whenever possible.
      2. The ratio between the volume of pollen and the volume of buffer A is critical for optimal disruption of pollen walls. The ratios described here lead to ~80-90% disruption efficiency. Adjust the ratio if pollen remains intact, or if the sample is heavily damaged as this will result in low yield of nuclei. More buffer increases breaking efficiency (and the risk of damage).
      3. To optimize the beating protocol we suggest to use two different frequencies and to run the beating protocol for 1, 1.5 and 2 min. 10 µl aliquots of the six samples should be mixed with 1 µl DAPI working solution and inspected at the microscope. 70-90% of all pollen grains should be broken and most of the released nuclei should float in the solution.
      4. Check the pH of the buffers if released nuclei are swollen and yellowish.
      5. Isolation of pollen nuclei takes about one hour.

  3. Fractionation of A. thaliana sperm and vegetative cell nuclei by fluorescence-activated cell sorting
    1. The cell sorter was equipped with a 70 µm nozzle and the sheath pressure was adjusted to 70 psi. For excitation, a 488 nm Coherent Sapphire Solid State 13 mW Laser was used. SYBR Green fluorescence was detected by a 530/30 nm band-pass filter. Cooling units were used to keep sample and fractionated nuclei at 4 °C during the whole procedure. Two labelled 15 ml Falcon tubes were placed into the device as collection tubes.
    2. The nuclei suspension was diluted with 0.5 volumes of buffer B and 250 µl were transferred to FACS tubes by filtering through 35 µm cell strainer caps.
    3. 5 µl of SYBR Green I was pipetted to one of the samples, mixed, and incubated for 5 min. Additional FACS tubes were kept on ice and stained with SYBR Green I just before sorting.
    4. Using the FACS Diva 6.1.2 software, a new FACS graph was prepared showing SSC-A (side-scatter area) on the y axis and FITC-A (area) on the x axis, both on a linear scale.
    5. To begin sorting, an unstained sample was analysed as negative control.
    6. Next, the SYBR Green I stained nuclei were loaded into the FACS device. Two fluorescent populations appear on the FACS plot, which were separately gated. For gating, a square was drawn around the densest part of the population. Outliers were ignored. Gating along the y-axis is not so critical whereas care has to be taken to avoid the area along the x-axis where both populations overlap to ensure purity of the sorted populations. To avoid sorting aggregates, like two adherent sperm nuclei, an auxiliary graph was set up that only showed the events gated in the first graph. This auxiliary plot with FITC-W (FITC width) on the y axis and FITC-A (FITC area) on the x axis reveals differences in the FITC-W value, which is higher in aggregates than in single nuclei. The two nuclear populations marked in Figure 2 B and D represent the desired nuclei and were gated for sorting. All events in this plot are gated. Once a sample tube was empty, a freshly stained sample tube was loaded into the FACS device.


      Figure 2. FACS plots of pollen nuclei during sorting. A. Wild-type A. thaliana (Col-0) pollen nuclei displayed as side scatter versus FITC-A (FITC area) of all events on linear scales. B. Auxiliary graph with FITC-W (FITC width) on the y axis and FITC-A (FITC area) on the x axis in linear scales that shows events gated in graph (A). The left population consists of sperm nuclei, the right population represents the vegetative nuclei. Both populations were gated for sorting. C. Dml-3 mutant (SALK_056440) A. thaliana pollen nuclei displayed as side scatter versus FITC-A (FITC area) of all events on linear scales. D. Auxiliary graph for graph (C).

    7. After sorting, a 10 µl aliquot of each population was stained with DAPI and inspected under a fluorescence microscope (as in step B7). Using 100x magnification, sperm and vegetative cell nuclei could be identified by differences in nucleus size, shape, and intensity of DAPI fluorescence (Figure 3). The average diameter (measured through the longest part) of sperm cell nuclei is 2.42 μm (which corresponds to Borges et al., 2012), the average diameter (measured through the longest part) of vegetative cell nuclei is 4.64 μm (Figure 3E). Sperm cell nuclei are regularly shaped (round or ellipsoid) whereas vegetative cell nuclei are highly lobed. DAPI stained sperm nuclei appear brighter than vegetative cell nuclei. The purity of each population was about 99%.
      Notes:
      1. The wrong FACS setting will result in no or only one population of nuclei in the FACS plot. Use the right nozzle and high pressure setting.
      2. Make sure to use a linear scale on both axes of the FACS plot.
      3. Use more SYBR green I if the nuclei populations in the FACS plot are very close to each other.
      4. If the population of vegetative cell nuclei is disappearing during the FACS run or if the yield is too low, the buffers and/or plants were too old.
      5. As the samples are not fixed, populations might move slightly over time and gating has to be monitored and adjusted if necessary.
      6. Fixing the sample before FACS (e.g., with formaldehyde) will result in large nuclear aggregates and is, therefore, not recommended.
      7. Depending on the amount of pollen used, FACS will take at least two hours.
      8. After the sort, rinse the system with diluted bleach to remove all SYBR Green I traces.
      9.  200 mg (~100 µl) wet mature pollen will result in ~1 x 106 sperm and 350,000 vegetative cell nuclei.
      10.  The expected 2:1 ratio of sperm to vegetative cell nuclei cannot be obtained, as vegetative cell nuclei have a more fragile morphology compared to sperm cell nuclei and can be lost. The average sperm versus vegetative cell nuclei ratio is 3:1.
      11. After the sort finished, we usually continued with purification. DNA extraction was performed as described in Schoft et al. (2011). RNA extraction was done according to Schoft et al. (2015). The samples can also be fixed (Schoft et al., 2015). Although we never tried, the samples should, in principle, survive flash freezing and storage at -80 °C.


        Figure 3. Microscopic images of sorted A. thaliana pollen nuclei. A-D. Nuclei were counterstained with DAPI and examined with the DAPI filter. Magnification 100x, size bar 2 μm. A and B Sperm cell nuclei. C and D Vegetative cell nuclei. E. Average diameter of sorted sperm and vegetative cell nuclei (measured through the longest part of the nuclei; length in μm; error bar: standard deviation).

Recipes

  1. Acid-washed glass beads
    1. Carefully pour glass beads into a 250 ml Erlenmeyer flask. Don't add more than 100 ml of glass beads.
    2. Cover the glass beads with nitric acid and shake the flask in an orbital shaker overnight. Use speed settings that allow the glass beads to move a bit.
    3. Discard the nitric acid and wash the glass beads with water until the pH is neutral.
    4. Remove the water and sterilize the glass beads in a drying oven at 180 °C for 4 h.
  2. Sucrose solution (working solution 9%, stock 45%)
    1. Dissolve 450 g sucrose in 1 L of water, autoclave and keep at room temperature.
    2. Dilute 1:5 with sterile water for working solution.
  3. Buffer A
    1. 1 M sorbitol, 7% ficol PM 400, 5 mM MgAc, 3 mM CaCl2, 5 mM EGTA, 50 mM Tris-HCl (pH 7.5), 100% Triton X100, glycerol.
    2. Mix 9.1 g sorbitol, 3.5 g Ficoll PM 400, 0.05 g Mg acetate, 0.022 g CaCl2, 0.095 g EGTA and 0.3 g Tris in 35 ml of sterile water.
    3. Adjust the pH to 7.5 with 1 M HCl.
    4. Add 1 ml Triton x100 (final concentration 2%) and 10 ml glycerol (final concentration 20%). Stored at 4 °C. The solution can be used for 3-4 weeks.
    5. Protease inhibitor cocktail (1:50 dilution) and PMSF (1:100 dilution) should be added just before use.
  4. Buffer B
    1. 15 mM Tris-HCl (pH 7.5), 2 mM Na2-EDTA, 0.5 mM spermine·4 HCl, 80 mM KCl, 20 mM NaCl, 2% Triton x100.
    2. Stock solutions:
      1. For 1 M Tris-HCl, dissolve 6.05 g in 30 ml water, adjust the pH to 7.5 with 5 M HCl and bring up to 50 ml with sterile water, autoclave;
      2. For 0.5 M EDTA, adjust the pH of 350 ml water to 8 with NaOH pellets and slowly add the salt while stirring, bring water up to 500 ml, autoclave;
      3. For 1 M spermine, dissolve 0.2 g in 10 ml water (final volume), sterile-filter;
      4. For 3 M KCl dissolve 22.365 g in 100 ml water (final volume), autoclave;
      5. For 5 M NaCl dissolve 29.22 g in 100 ml water (final volume), autoclave.
      6. For 5 ml working solution mix 75 µl Tris-HCl, 20 µl EDTA, 25 µl spermine, 133 µl KCl, 20 µl NaCl and 100 µl Triton X-100, bring up to 5 ml with sterile water.
      7. Stored at 4 °C.
      8. Protease inhibitor cocktail (1:50 dilution) and PMSF (1:100 dilution) should be added just before use.
  5. Protease inhibitor stock solution
    For 50x concentration, one tablet is dissolved in 1 ml water
  6. DAPI (working solution 10 µg/ml; stock 2 mg/ml)
    1. Add 5 ml sterile water to 10 mg of DAPI powder for the stock solution
    2. Dilute stock solution 1:200 with sterile water for the working solution (“DAPI in water”)
    3. Protect from light
    4. Stored at -20 °C

Acknowledgments

This protocol is based on previously published work (Schoft et al., 2009; Schoft et al., 2011; Ibarra et al., 2012; Schoft et al., 2015). We thank the BioOptics Facility of the Research Institute of Molecular Pathology, Vienna, for setting up and optimizing FACS. We thank Gerald Schmauss for measuring sizes of sorted A. thaliana pollen nuclei. We thank Hisashi Tamaru for his support. This work was supported by Austrian Science Fund (FWF) Grants P21389-B03 and P24918-B21.

References

  1. Boyes, D. C., Zayed, A. M., Ascenzi, R., McCaskill, A. J., Hoffman, N. E., Davis, K. R. and Gorlach, J. (2001). Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13(7): 1499-1510.
  2. Borges, F., Gardner, R., Lopes, T., Calarco, J. P., Boavida, L. C., Slotkin, R. K., Martienssen, R. A. and Becker, J. D. (2012). FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods 8(1): 44.
  3. Honys, D. and Twell, D. (2003). Comparative analysis of the Arabidopsis pollen transcriptome. Plant Physiol 132(2): 640-652.
  4. Ibarra, C. A., Feng, X., Schoft, V. K., Hsieh, T. F., Uzawa, R., Rodrigues, J. A., Zemach, A., Chumak, N., Machlicova, A., Nishimura, T., Rojas, D., Fischer, R. L., Tamaru, H. and Zilberman, D. (2012). Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337(6100): 1360-1364.
  5. Johnson-Brousseau, S. A. and McCormick, S. (2004). A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes. Plant J 39(5): 761-775.
  6. Schoft, V. K., Chumak, N., Bindics, J., Slusarz, L., Twell, D., Kohler, C. and Tamaru, H. (2015). SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content. Plant Reprod 28(1): 61-72.
  7. Schoft, V. K., Chumak, N., Choi, Y., Hannon, M., Garcia-Aguilar, M., Machlicova, A., Slusarz, L., Mosiolek, M., Park, J. S., Park, G. T., Fischer, R. L. and Tamaru, H. (2011). Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte. Proc Natl Acad Sci U S A 108(19): 8042-8047.
  8. Schoft, V. K., Chumak, N., Mosiolek, M., Slusarz, L., Komnenovic, V., Brownfield, L., Twell, D., Kakutani, T. and Tamaru, H. (2009). Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin. EMBO Rep 10(9): 1015-1021.

简介

植物和动物生物学的一个主要议题是有性繁殖。因此,分离和研究生殖细胞和附属细胞是非常有意义的。开花植物拟南芥(Arabidopsis thaliana)( A。thaliana )的雄性配子体是两个减数分裂后有丝分裂的产物。每个成熟花粉粒由包含在营养细胞(非生殖伴侣细胞)内的两个精细胞组成。坚硬的花粉壁及其特殊的嵌套结构使得难以分别研究花粉细胞。在这里,我们描述一个简单而有效的方法分割。拟南芥精子和营养细胞核通过荧光激活细胞分选(FACS)。我们的方案基于用SYBR Green I染色的精子和营养细胞核的荧光强度的差异.100植物产生约1×10 6个精子和350,000个营养细胞核。该方法可用于纯化各种A的花粉核。拟南芥野生型种质和突变株系,并且原则上可适用于其它植物物种的花粉。

关键字:荧光染料掺入法, 荧光激活细胞分选(FACS), 拟南芥, 花粉, 雄配子体

材料和试剂

  1. 塑料制品/耗材
    1. 15ml聚丙烯管(SARSTEDT AG& Co,目录号:62554502)
    2. 50ml聚丙烯管(SARSTEDT AG& Co,目录号:62548004)
    3. 100μm的尼龙网(Biologix Group Limited,目录号:151100)
    4. 40μm尼龙网(Biologix Group Limited,目录号:151040)
    5. 250ml离心管(Bartelt,目录号:9.315721)
    6. 1.5ml和2ml微升反应管
    7. 注射器过滤器0.2μm(Bartelt,目录号:9055511)
    8. 具有细胞过滤帽的BD Falcon 12x75mm管(BD Biosciences,目录号:352235)
      注意:目前,它是"康宁公司,目录号:352235"。
    9. 一次性皮下注射针,直径为0.45×25 mm(B.Braun Melsungen AG,Braun Petzold,目录号:4657683)
    10. 移液管吸头(1,000μl,200μl,20μl,2μl)
    11. 酸洗玻璃珠(见配方)

  2. 玻璃器皿
    1. 玻璃珠0.4-0.6mm(Sartorius AG,??目录号:BBI8541701)
    2. 250ml锥形瓶
    3. 玻璃管
    4. 玻璃滑槽
    5. 封面

  3. 植物材料
    1. 100开花的野生型拟南芥植物(Col-0登记号 和dml-3突变体作为实例)(Arabidopsis,目录号: SALK_056440)

  4. 化学品
    1. D(+) - 蔗糖用于分析(AppliChem GmbH,目录号:131621.0914)
    2. DAPI(4',6-二脒基-2-苯基吲哚,二盐酸盐)(Life Technologies,Invitrogen TM,目录号:D1306)
      注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:D1306"。
    3. SYBR Green I核酸染色原液(Life Technologies,目录号:S7567)
      注意:目前,"Thermo Fisher Scientific,Invitrogen TM ,目录号:S7567"。
    4. 苯基甲磺酰氟(Sigma-Aldrich,目录号:93482)
    5. 完全,无EDTA蛋白酶抑制剂混合物片剂(Roche Diagnostics,目录号:11873580001)
    6. 氯化钙二水合物(AppliChem GmbH,目录号:A3587)
    7. 乙二胺四乙酸二钠盐二水合物(Carl Roth GmbH + Co.,目录号:8043)
    8. 乙二醇 - 双(2-氨基乙醚)-N,N,N,N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
    9. Ficoll?PM 400(Sigma-Aldrich,目录号:F4375)
    10. 甘油(Sigma-Aldrich,目录号:G5516)
    11. 醋酸镁四水合物(Sigma-Aldrich,目录号:M5661)
    12. 氯化钠(EMD Millipore Corporation,目录号:106404)
    13. 氯化钾(Sigma-Aldrich,目录号:P3911)
    14. D-山梨醇(Sigma-Aldrich,目录号:S6021)
    15. 精胺[N,N'-双 - (3-氨基丙基)-1,4-二氨基丁烷](AppliChem GmbH,目录号:A0672.0005)
    16. TritonX-100(Sigma-Aldrich,目录号:234729)
    17. Tris-base(AppliChem GmbH,目录号:A1086)
    18. 硝酸65%(Carl Roth GmbH + Co.,目录号:X898.1)
    19. 盐酸发烟37%(Carl Roth GmbH + Co.,目录号:4625.1)
    20. 氢氧化钠颗粒(AppliChem GmbH,目录号:A3910)
    21. 超纯水(MilliQ)(电导率>18MΩ<1cm -1 -1 )

  5. 缓冲区
    1. BD FACSFlow鞘液(BD Bioscience,目录号:342003)
    2. 蔗糖溶液(见配方)
    3. 缓冲区A(参见配方)
    4. 缓冲液B(参见配方)
    5. 蛋白酶抑制剂储备溶液(参见配方)
    6. DAPI储备溶液(见配方)

设备

  1. 仪器
    1. 带有JA-10转子的Beckman Coulter Avanti J-26XP离心机
    2. 台式离心机
    3. 轨道振动器(VWR International,目录号:89032102)
    4. 干燥炉(EHRET GmbH,目录号:3108)
    5. Bead-beater(Retsch GmbH,型号:MM301)
    6. 带有20x,40x和100x物镜和DAPI滤光片的荧光显微镜
    7. 流式细胞仪:具有70μm喷嘴的FACS Aria I(BD Biosciences) ?70psi护套压力,以及488nm相干蓝宝石固态13 mW激光器和用于检测的530/30nm带通滤波器 的SYBR绿色I

  2. 一般实验室设备
    1. 烧杯
    2. 250ml锥形烧瓶
    3. 玻璃管
    4. 锋利的剪刀
    5. 镊子
    6. 空气移液管
    7. 冰容器

软件

  1. 流式细胞术软件(BD Biosciences,型号:FACS Diva 6.1.2)

程序

  1. 释放 A。如先前所述(Honys和Twell,2003; Johnson-Brousseau和McCormick,2004; Schoft等人,2009),其具有修改]
    (来自花序的
    1. 将?100株植物(拟南芥)在16小时光照/8小时黑暗条件下生长5周 循环在22℃。植物的发育阶段对应于6.10至 ?6.50根据表2在Boyers等人(2001)。
    2. 通过用锋利的剪刀剪切将所有花序收集在烧杯中(置于冰上)。
    3. 将9%冰冷的蔗糖溶液倒在收集的上 花序直到被覆盖。花序是 剧烈旋转一分钟以释放成熟花粉粒
    4. 将悬浮液通过100μm尼龙网过滤到置于冰上的250ml离心管中
    5. 花粉颗粒通过在4℃下以1350×g离心10分钟浓缩。
    6. 将上清液倒回到花序上,并再次重复步骤3-5
    7. 将花粉沉淀重悬于冰冷的缓冲液A中至最终 体积为10ml,并通过40μm的尼龙网过滤到15ml 猎鹰管。
    8. 花粉颗粒通过在4℃以800×g离心10分钟沉淀
    9. 花粉颗粒再次用1ml缓冲液A洗涤 通过在37℃下离心收集在无菌的1.5ml管中 在桌上型离心机中在4℃下5分钟,5分钟。上清液 舍弃。
      注意:
      1. 收集花序和释放花粉需要约2-3小时。
      2. 沉淀的花粉可以立即使用或快速使用 在液氮中冷冻并在-80℃保存几年。
      3. 100只拟南芥植物(Col-0生态型)产生约100μl花粉。 花粉粒体体积通过填充相同的空管来估计 用水达到与花粉相同的水平并测量水 体积用空气置换移液管。此金额可能会有所不同 ?加入,生长条件和植物年龄。

  2. 隔离 A。 thaliana 花粉核的成熟花粉
    1. 10-20个无菌2ml离心管中各装入1.8g 酸洗玻璃珠。未使用的管可以在室温下储存 并稍后使用。
    2. 将收获的花粉与缓冲液A混合 补充有PMSF和蛋白酶抑制剂 花粉与缓冲液A比率:
      在花粉颗粒体积80μl下 比例为1:3(花粉:缓冲液A)的比例在80μl和150μl之间 ?使用1:3.5,高于150μl,使用1:4的比例 花粉颗粒在缓冲液A中的再悬浮
    3. 花粉粒 将悬浮液小心地移液到装有2ml离心管中 ?玻璃珠(在步骤B1中制备)以50μl等分试样
    4. 花粉 通过放置填充的2ml管对颗粒进行珠磨 与花粉悬浮液和玻璃珠在Retsch球磨机和, 随后,在15Hz下运行打浆方案1.5分钟。
    5.  用新鲜的管子将三个孔冲入管的底部 针。小心地切下盖子,并放置2ml管 也取下了其盖子的无菌1.5ml管的顶部。的 然后将两个管放入用于离心的玻璃管中 将在1.5ml管中回收核悬浮液 (或者,可以使用15ml Falcon管)(图1A)。
    6. 将玻璃管在4℃下以800×g离心10分钟。 2毫升 弃去含有玻璃珠的管并且除去核 将悬浮液合并到单个无菌的1.5ml离心管中
    7. 将10μl花粉核悬浮液与1μlDAPI混合 水并吸移到载玻片上。仔细放置盖玻片 ?在样品顶部。花粉核释放的效率和质量 的细胞核用荧光显微镜使用20x和40x评估 ?目标和DAPI通道[DAPI的激发/发射(nm): 358/461](图1B和C)
         A


      图1.评估 A。 thaliana 花粉核释放效率。 A.三管设置前后 离心。 B.A的显微镜图像。拟南芥野生型(Col-0) 花粉在珠打之前,DAPI:用DAPI染色的花粉检查 DAPI过滤器。 40x放大。尺寸条:25μm。 C.A的显微镜图像。拟南芥野生型花粉(Col-0)。箭头:(1) 完整花粉,(2)细胞核仍附着于细胞质,(3)空花粉 壳,(4)释放营养核。 DAPI:DAPI染色的花粉 用DAPI过滤器检查。 40x放大。尺寸栏:25μm

      注意:
      1. 离心管应尽可能保持在4°C。
      2. 花粉的体积与缓冲液A的体积之间的比率为 ?对于花粉壁的最佳破坏至关重要。所描述的比率 这里导致?80-90%的破坏效率。如果花粉调整比率 保持完好,或者如果样品被严重损坏,因为这将导致 在低产率的核。更多缓冲区提高打破效率(和 损害的风险)。
      3. 为了优化跳动协议,我们建议 使用两个不同的频率和运行的跳动协议为1, 1.5和2分钟。 10个等份的六个样品应该与1混合 μlDAPI工作溶液,并在显微镜下检查。 70-90%的 花粉粒应该破碎,大多数释放的核应该 float在解决方案中。
      4. 如果释放的细胞核溶胀并变黄,请检查缓冲液的pH值。
      5. 花粉核的分离需要大约一小时。

  3. 分数A。 thaliana 精子和营养细胞核通过荧光激活细胞分选
    1. 细胞分选仪装配有70μm喷嘴和鞘压力 ?调节至70psi。对于激发,488nm相干蓝宝石 使用固态13mW激光器。检测到SYBR绿色荧光 通过530/30nm带通滤波器。使用冷却单元保持样品 并在整个过程中在4℃分离核。两个标签 将15ml Falcon管置于装置中作为收集管
    2. 用0.5体积的缓冲液B稀释核悬浮液 通过用35μm细胞过滤将250μl转移到FACS管中 过滤器帽
    3. 将5μl的SYBR Green I移液至其中一个 样品,混合,并孵育5分钟。保存另外的FACS管 ?在冰上,并在分拣前用SYBR Green I染色
    4. 使用 FACS Diva 6.1.2软件,制备了新的FACS图 在y轴上的SSC-A(侧向散射区域)和在x轴上的FITC-A(面积) ?两者在线性标度上。
    5. 为了开始分选,将未染色的样品作为阴性对照进行分析
    6. 接下来,将SYBR Green I染色的细胞核装载到FACS中 设备。两个荧光种群出现在FACS图上 单独门控。对于门控,在最密集部分周围画一个正方形 ?的人口。异常值被忽略。沿y轴的门是 不是那么关键,而必须小心避免沿着该地区 ?x轴,其中两个群体重叠以确保分选的纯度 人口。避免分选聚集体,如两个粘附精子 核,设置了仅显示门控的辅助图 在第一个图中。这个辅助图在y上具有FITC-W(FITC宽度) ?轴和FITC-A(FITC面积)在x轴上显示的差异 FITC-W值,其在聚集体中高于单核。的 图2B和D中标记的两个核群体代表所需的 ?核并进行门排序。该图中的所有事件都是门控的。 一旦样品管为空,装载新鲜染色的样品管 进入FACS设备

      图2.花粉核的FACS图 A.野生型。 thaliana (Col-0)花粉核显示为 侧散射对FITC-A(FITC面积)的线性尺度上的所有事件。 B.在y轴和FITC-A上具有FITC-W(FITC宽度)的辅助图 (FITC区域)在x轴上的线性刻度显示事件门控 图(A)。左边的人群由精子细胞核组成,右边 群体代表营养核。两个群体都是门控的 ?用于排序。 C.Dml-3突变体(SALK_056440)。 thaliana 花粉核 显示为所有事件的侧散射对FITC-A(FITC面积) 线性尺度。 D.图(C)的辅助图。

    7. 后 分选,每个群体的10μl等分试样用DAPI染色 在荧光显微镜下检查(如步骤B7中)。使用100x 放大,精子和营养细胞核可以鉴定 DAPI荧光的核大小,形状和强度的差异 (图3)。平均直径(通过最长部分测量)为 精子细胞核是2.42μm(其对应于Borges等人,2012), ?植物的平均直径(通过最长部分测量) 细胞核是4.64μm(图3E)。精子细胞核定期 (圆形或椭圆形),而营养细胞核高度 lobed。 DAPI染色的精子核显示比营养细胞更亮 核。每个群体的纯度约为99%。
      注意:
      1. 错误的FACS设置将导致没有或只有一个人口 细胞核。使用正确的喷嘴和高压设置。
      2. 确保在FACS图的两个轴上使用线性刻度。
      3. 如果FACS图中的细胞核群体彼此非常接近,请使用更多的SYBR green I。
      4. 如果营养细胞核的群体在消失期间 FACS运行或如果产率太低,则缓冲液和/或植物 太旧了。
      5. 由于样本不固定,人群可能移动 略微随时间和门控必须监测和调整如果 必要。
      6. 在FACS之前固定样品(例如 甲醛)将导致大的核聚集体,因此, ?不推荐。
      7. 根据使用的花粉量,FACS至少需要两个小时。
      8. 分拣后,用稀释的漂白剂冲洗系统以清除所有SYBR Green I痕迹。
      9. 200mg(?100μl)湿成熟花粉将导致?1×10 6个
        精子和35万个营养细胞核。
      10. 精子与营养细胞核的预期的2:1比率不能 因为营养细胞核具有更脆弱的形态 相比精子细胞核和可以丢失。平均精子与 营养细胞核比例为3:1。
      11. 分类完成后,我们 通常继续纯化。进行DNA提取 描述于Schoft等(2011)。 RNA提取根据 Schoft等人(2015)。样品也可以是固定的(Schoft等人, 2015)。虽然我们从未试过,样品应该,原则上, 在-80℃下快速冷冻并储存。



        图3.显微镜 ?排序的 A的图像。 thaliana 花粉核。 A-D。核 用DAPI复染色并用DAPI滤光片检查。 放大倍数100x,尺寸条2μm。 A和B精子细胞核。 C和D. 营养细胞核。分拣精子的平均直径和 营养细胞核(通过核的最长部分测量; ?长度(μm)误差棒:标准偏差)。

食谱

  1. 酸洗玻璃珠
    1. 小心地将玻璃珠倒入250ml锥形烧瓶中。不要加入超过100毫升的玻璃珠。
    2. 用硝酸覆盖玻璃珠,并在烧瓶中摇动 轨道振荡器过夜。使用允许玻璃珠的速度设置 移动一点。
    3. 弃去硝酸,用水洗涤玻璃珠直到pH值为中性
    4. 除去水,在180℃的干燥炉中对玻璃珠灭菌4小时。
  2. 蔗糖溶液(工作溶液9%,原料45%)
    1. 将450g蔗糖溶解在1L水中,高压灭菌并保持在室温
    2. 稀释1:5用无菌水稀释工作溶液。
  3. 缓冲区A
    1. 1μM山梨醇,7%ficol PM 400,5mM MgAc,3mM CaCl 2,5mM EGTA,50mM Tris-HCl(pH 7.5),100%Triton X100,甘油。 >
    2. 混合9.1g山梨醇,3.5g Ficoll PM 400,0.05g乙酸镁,0.022g CaCl 2,0.095g EGTA和0.3g Tris的35ml无菌水溶液。
    3. 用1M HCl调节pH至7.5
    4. 加入1ml Triton x100(终浓度2%)和10ml甘油 (终浓度20%)。储存于4°C。该解决方案可用于 3-4周。
    5. 应在使用前加入蛋白酶抑制剂混合物(1:50稀释)和PMSF(1:100稀释)。
  4. 缓冲区B
    1. 15mM Tris-HCl(pH7.5),2mM Na 2 EDTA,0.5mM精胺·HCl,80mM KCl,20mM NaCl,2%Triton×100。
    2. 库存解决方案:
      1. 对于1M Tris-HCl,溶解6.05g在30ml水中,调节pH至 7.5用5M HCl,并用无菌水加热至50ml,高压灭菌;
      2. 对于0.5M EDTA,用NaOH调节350ml水的pH至8 颗粒并缓慢加入盐,同时搅拌,使水达500 ml,高压灭菌;
      3. 对于1M精胺,将0.2g溶于10ml水(最终体积),无菌过滤器中;
      4. 对于3M KCl,在100ml水(最终体积)中溶解22.365g,高压灭菌;
      5. 对于5M NaCl溶于29.22g在100ml水(最终体积)中,高压灭菌
      6. 对于5ml工作溶液混合物75μlTris-HCl,20μlEDTA,25μl 精胺,133μlKCl,20μlNaCl和100μlTriton X-100,达到5 ml用无菌水
      7. 储存在4°C。
      8. 应在使用前加入蛋白酶抑制剂混合物(1:50稀释)和PMSF(1:100稀释)。
  5. 蛋白酶抑制剂储备液
    对于50倍浓度,将一片溶解在1ml水中
  6. DAPI(工作溶液10μg/ml;储备2mg/ml)
    1. 加入5毫升无菌水至10毫克DAPI粉末的储备溶液
    2. 用工作溶液("DAPI水中")的无菌水稀释储备溶液1:200
    3. 避光
    4. 储存于-20°C

致谢

该协议基于以前公开的工作(Schoft等人,2009; Schoft等人,2011; Ibarra等人,2012年) ; Schoft等人,2015)。我们感谢维也纳分子病理学研究所的BioOptics设施,用于设置和优化FACS。我们感谢Gerald Schmauss测量排序的大小。 thaliana 花粉核。我们感谢Hisashi Tamaru的支持。这项工作得到奥地利科学基金(FWF)拨款P21389-B03和P24918-B21的支持。

参考文献

  1. Boyes,D.C.,Zayed,A.M.,Ascenzi,R.,McCaskill,A.J.,Hoffman,N.E.,Davis,K.R.and Gorlach,J。(2001)。 拟南芥的基于生长阶段的表型分析: :高通量的模型功能性基因组学。植物细胞 13(7):1499-1510。
  2. Borges,F.,Gardner,R.,Lopes,T.,Calarco,J.P.,Boavida,L.C。,Slotkin,R.K.,Martienssen,R.A。和Becker,J.D。(2012)。 基于FACS的拟南芥小孢子,精细胞和营养核的纯化。 植物方法 8(1):44.
  3. Honys,D。和Twell,D。(2003)。 拟南芥花粉转录组的比较分析 > Plant Physiol 132(2):640-652。
  4. Ibarra,CA,Feng,X.,Schoft,VK,Hsieh,TF,Uzawa,R.,Rodrigues,JA,Zemach,A.,Chumak,N.,Machlicova,A.,Nishimura,T.,Rojas, ,Fischer,RL,Tamaru,H。和Zilberman,D。(2012)。 植物伴侣细胞中的活性DNA去甲基化加强了配子中的转座子甲基化。 科学 337(6100):1360-1364。
  5. Johnson-Brousseau,S.A。和McCormick,S。(2004)。 有用于表征拟南芥花粉突变体和配子体表达的方法纲要基因。植物J 39(5):761-775。
  6. Schoft,V.K.,Chumak,N.,Bindics,J.,Slusarz,L.,Twell,D.,Kohler,C.and Tamaru,H。 SYBR绿色激活的拟南芥花粉核的排序基于不同的DNA/RNA内容 植物Reprod 28(1):61-72
  7. Schoft,VK,Chumak,N.,Choi,Y.,Hannon,M.,Garcia-Aguilar,M.,Machlicova,A.,Slusarz,L.,Mosiolek,M.,Park,JS,Park,GT,Fischer ,RL和Tamaru,H。(2011)。 DEMETER DNA糖基化酶在拟南芥雄性配子体中的功能。 Natl Acad Sci USA 108(19):8042-8047。
  8. Schoft,V.K.,Chumak,N.,Mosiolek,M.,Slusarz,L.,Komnenovic,V.,Brownfield,L.,Twell,D.,Kakutani,T.and Tamaru, 在组成型异染色质的去扩缩时诱导RNA指导的DNA甲基化。 EMBO Rep 10(9):1015-1021。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chumak, N., Mosiolek, M. and Schoft, V. K. (2015). Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS. Bio-protocol 5(22): e1664. DOI: 10.21769/BioProtoc.1664.
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