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In vitro Detection of Neutrophil Traps and Post-attack Cell Wall Changes in Candida Hyphae
念珠菌菌丝中嗜中性粒细胞诱捕和攻击后细胞壁变化的体外检测   

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Abstract

In this protocol we describe how to visualize neutrophil extracellular traps (NETs) and fungal cell wall changes in the context of the coculture of mouse neutrophils with fungal hyphae of Candida albicans. These protocols are easily adjusted to test a wide array of hypotheses related to the impact of immune cells on fungi and the cell wall, making them promising tools for exploring host-pathogen interactions during fungal infection.

Keywords: Fungi(真菌), C. albicans(白色念珠菌), NETs(NETs), Host-pathogen interactions(宿主 - 病原体相互作用), Cell wall(细胞壁), sDectin-1-Fc(sDectin-1-Fc)

Background

C. albicans is a polymorphic opportunistic yeast and neutrophils are immune cells critical for defense against this and other fungal pathogens (Brown et al., 2012; Lionakis and Netea, 2013). NETs are a potential defense mechanism that can be deployed against pathogens and it has been suggested that they are preferentially deployed against microbial cells such as C. albicans hyphae that are too large to phagocytose (Urban et al., 2006; Bruns et al., 2010; Branzk et al., 2014; Rohm et al., 2014). NETs have been shown to contain a number of components including myeloperoxidase, extracellular DNA and citrullinated histones (Amulic et al., 2012; Branzk and Papayannopoulos, 2013). For positive identification of NETs, the standard in both in vitro and in vivo experiments includes staining for and demonstrating the colocalization of these markers. While the exact contribution of NETs to defense against C. albicans infection is not well understood, our group has demonstrated they can provoke stress responses and cell wall rearrangement in C. albicans hyphae. Specifically, NET attack results in greater chitin deposition and β-glucan exposure as shown schematically in Figure 1. These polysaccharides normally lie underneath the mannan layer, and their exposure can change immune recognition (Perez-Garcia et al., 2011). The basic assays described here were used extensively to probe this subject by our group (Hopke et al., 2016). While outlined here for the purpose of detecting NETs and fungal cell wall changes, this protocol is easily tweaked to leverage many combinations of chemical inhibitors, transgenic or knockout fungal strains or mouse neutrophils and other staining targets to test a wide array of hypotheses (Hopke et al., 2016). This protocol therefore represents a promising method to further elucidate the impact immune cells have on the C. albicans cell wall, its stress response and the importance of altered epitope exposure to host defense against fungal infection.


Figure 1. Schematic of cell wall organization pre- and post-neutrophil attack. Under homeostatic conditions, hyphal cell wall is composed of three main polysaccharide components (mannan, β-glucan and chitin) but most chitin and β-glucan are inaccessible for recognition because it lies beneath the mannan layer. Post-attack there is loss of cell wall mannoprotein and increased levels of chitin. These changes lead to greater surface recognition of β-glucan and perhaps also chitin.

Materials and Reagents

  1. Protective gloves and lab coat
  2. Flint glass culture tubes 16 x 150 mm (VWR, catalog number: 60825-435 )
  3. 50 ml conical tubes (Corning, Falcon®, catalog number: 352098 )
  4. Fisherbrand 100 x 20 mm Petri dishes (Fisher Scientific, catalog number: FB0875711Z )
  5. 10 ml syringe (BD, catalog number: 301604 )
  6. 25 G 5/8 needle (BD, catalog number: 305122 )
  7. 70 µm cell strainers (Corning, Falcon®, catalog number: 352350 )
  8. 1.7 ml microcentrifuge tubes
  9. Plain microscope slides (VWR, catalog number: 48300-025 )
  10. Kim-KapTM Disposable closures; 16 mm (Kimble Chase Life Science and Research Products, catalog number: 7366316 )
  11. Miltenyi columns (Miltenyi Biotec, catalog number: 130-021-101 )
  12. WT-FarRed670 strain of C. albicans (Hopke et al., 2016)
  13. WT-GFP strain (Wheeler et al., 2008)
  14. C57BL/6J mice, female 6-10 weeks old (THE JACKSON LABORATORIES, catalog number: 000664 )
  15. Glycerol (EMD Millipore, catalog number: GX0185-5 )
  16. Anti-Ly6G biotin antibody (Thermo Fisher Scientific, eBioscience, catalog number: 13-5931-85 )
  17. Anti-biotin magnetic beads (Miltenyi Biotec, catalog number: 130-090-485 )
  18. Trypan blue (Lonza, catalog number: 17-942E )
  19. FluoReporter® Cell-Surface Biotinylation Kit (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: F20650 )
  20. Alexa Fluor 647 conjugated streptavidin (Jackson ImmunoResearch, catalog number: 016-600-084 )
  21. Anti-myeloperoxidase (MPO) (R&D Systems, catalog number: AF3667 )
  22. Anti-histone H3 (citrulline R2+R8+R17) (Abcam, catalog number: ab5103 )
  23. Sytox Green (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S7020 )
  24. Calcofluor white/Fluorescent brightener 28 (Sigma-Aldrich, catalog number: F3543 )
  25. Donkey anti-goat IgG Cy3 (Jackson ImmunoResearch, catalog number: 705-165-147 )
  26. Donkey anti-rabbit IgG Cy3 (Jackson ImmunoResearch, catalog number: 711-165-152 )
  27. sDectin-1-Fc (produced in house according to [Graham et al., 2006])
  28. Donkey anti-human IgG Cy3 (Jackson ImmunoResearch, catalog number: 709-165-149 )
  29. Nail polish
  30. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
  31. Sodium phosphate, dibasic anhydrous (Na2HPO4) (Fisher Scientific, catalog number: S374-500 )
  32. Potassium phosphate, monobasic anhydrous (KH2PO4) (Fisher Scientific, catalog number: P285-500 )
  33. Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
  34. BD Bacto peptone (BD, BactoTM, catalog number: 211677 )
  35. BD Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
  36. Dextrose (Fisher Scientific, catalog number: D-163 )
  37. BD Bacto agar (BD, BactoTM, catalog number: 214014 )
  38. Heat inactivated fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  39. Probumin® bovine serum albumin diagnostic grade powder (EMD Millipore, catalog number: 820451 )
  40. RPMI, with 25 mM HEPES and L-glutamine (Lonza, catalog number: 12-115F )
  41. Ammonium chloride (NH4Cl) (EMD Millipore, catalog number: AX12701 )
  42. Crystal violet (Sigma-Aldrich, catalog number: C0775 )
  43. Acetic acid (Fisher Scientific, catalog number: A38-212 )
  44. Tris base (AMRESCO, catalog number: 0497 )
  45. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-500 )
  46. PBS, pH 7.2 (see Recipes)
  47. PBS + Na2CO3, pH 8 (see Recipes)
  48. YPD broth (see Recipes)
  49. PBS + 5% FBS (see Recipes)
  50. PBS + 2% FBS (see Recipes)
  51. PBS + 2% BSA (see Recipes)
  52. RPMI + 5% FBS (see Recipes)
  53. Tris-HCl, pH 8.0 (see Recipes)
  54. TAC buffer (see Recipes)
  55. Turks solution (see Recipes)

Equipment

  1. Roller drum (Eppendorf, New Brunswick Scientific) and incubators (VWR)
  2. Hemacytometer for cell counting (Hausser Scientific, catalog number: 1492 )
  3. Spectrophotometer (Eppendorf; Biophotometer)
  4. Centrifuge with adaptors for 15 and 50 ml conical tubes (Thermo Fisher Scientific, model: SorvallTM LegendTM RT )
  5. AutoMACs separation system (Miltenyi Biotec, model: autoMACS® Pro Separator )
  6. Zeiss Axiovision fluorescence microscope (Zeiss; custom build)
  7. Dissection kit (VWR, catalog number: 631-0616 )
  8. pH meter (Fisher Scientific, model: accumetTM Excel XL15 )
  9. Microcentrifuge for 1.7 ml tubes (Eppendorf, model: 5424 )
  10. Autoclave
  11. 500 ml bottle (VWR, catalog number: 89000-233 )
  12. Water bath

Software

  1. AxioVision Rel48 software (Zeiss: https://www.zeiss.com/microscopy/us/downloads/axiovision-downloads.html)
  2. Photoshop (Adobe Systems, San Jose, CA)

Procedure

  1. Growth of Candida albicans hyphae (3 days; see Figure 2A for a schematic of the procedure).


    Figure 2. Schematic of the first three steps of the protocol

    1. Streak the appropriate strain of C. albicans to single colonies from a frozen 25% glycerol stock onto YPD agar (see Recipes) and incubate at 37 °C overnight. For these experiments, we use the WT-FarRed670 strain of C. albicans (Genotype: PENO1::PENO1-FarRed670-NATR; [Hopke et al., 2016]).
    2. The next day pick a single colony and transfer into 5 ml of YPD liquid (see Recipes). We put the 5 ml in 16 x 150 mm flint glass tubes with caps and incubate at 30 °C on a roller drum overnight. Harvest the YPD culture and count the yeast using a hemocytometer.
    3. Dilute into 30 ml of RPMI at a concentration of 2.5 x 106 cells/ml and divide into 5 ml aliquots. Incubate at 30 °C on a roller drum overnight. Pool the RPMI cultures into a 50 ml conical tube and spin down at 3,000 x g for 5 min. Remove most of the supernatant (so only about 5 ml remains) and then flick to resuspend the pellet in remaining liquid. Due to the morphology of the hyphae, you cannot use a hemocytometer to determine cell number, instead we used the spectrophotometer to estimate the concentration of the hyphae. An OD600 of 1 is equivalent to 1 x 107 cells/ml. While quantification of hyphae by OD600 is not as accurate as for quantifying yeast, we found it to provide reproducible results. Adjust to 3 x 108 cells/ml to use as a stock solution in the assay.
      Note: You will have a highly concentrated number of hyphae in a small volume, so it is best to do a dilution before finding the OD600 to ensure you fall within a readable range and to conserve cells. Our cuvettes usually take 1 ml, so we take 100 µl hyphae and add it to 900 µl PBS. PBS will then serve as your blank control.
  2. Isolation of mouse bone marrow (1-2 h; see Figure 2B for a schematic of the procedure; for a detailed video protocol see [Swamydas and Lionakis, 2013])
    1. Euthanize two C57BL/6J female mice of 6-10 weeks of age via CO2 inhalation followed by cervical dislocation. Dissect the hind legs to acquire the femurs and tibiae. Remove all the flesh and transfer into a Petri dish with PBS + 5% FBS (see Recipes). 
    2. Fill a 10 ml syringe with the PBS + 5% FBS and attach a 25 G 5/8 needle. Insert the needle into the bones and expel the liquid from the syringe to force out the bone marrow into the Petri dish (you can clip the ends of the bones if it is difficult to insert the needle).
    3. When all the bone marrow has been released into the Petri dish, pass it through a 70 µm cell strainer and into a 50 ml conical. Centrifuge at 300 x g for 3 min.
    Notes:
    1. It is best to expel the bone marrow in a Petri dish that has no remnants of soft tissue. The tissue will clog the needle. If necessary, you can finish cleaning the bones in one Petri dish and then move the bones to another one with PBS + 5% FBS before expelling the marrow.
    2. All animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animals were euthanized by carbon dioxide inhalation. Infected animals were monitored twice daily for signs of infection and morbid animals were euthanized. The UMaine IACUC/Ethics Committee approved this protocol.
  3. Isolation of neutrophils from mouse bone marrow via AutoMACs (1.5-2 h; see Figure 2C for a schematic of the procedure)
    1. Discard the supernatant and resuspend the pellet by flicking the conical tube. Add 3 ml of Tris/ammonium chloride (TAC) buffer (see Recipes) and incubate at room temperature for 1 min to lyse any red blood cells. After the incubation, dilute to 30 ml total with PBS + 2% FBS. Take 10 µl of the bone marrow solution and add it to 40 µl Turks solution (see Recipes). Count the cells on a hemocytometer.
    2. Centrifuge the bone marrow solution at 300 x g for 3 min. Discard the supernatant and flick resuspend the pellet in PBS + 2% FBS at a concentration of 1 x 108 cells/ml. Incubate with anti-Ly6G biotin antibody (stock at 0.5 mg/ml) at 1 µl antibody per 100 µl bone marrow on ice for 15 min. Wash by centrifuging at 300 x g for 3 min and discarding the supernatant. Resuspend in 10 ml PBS + 5% FBS then centrifuge again. Discard the supernatant and resuspend again in 10 ml PBS + 5% FBS. Incubate with anti-biotin magnetic beads (10 µl/1 x 107 cells) on ice for 30 min. Agitate periodically throughout the incubation. Wash once with 10 ml PBS + 5% FBS as outlined above. Resuspend the pellet in 1 ml of PBS + 5% FBS.
      Note: During this time turn on the AutoMACS and run the CLEAN cycle.
    3. Once finished put clean 50 ml conical tubes in the positive and negative ports. Put the bone marrow at the intake port and run the positive selection program. When all but 0.2 ml of the volume has been taken up the intake, add 1 ml PBS + 2% FBS. This ensures that all of the residual sample is loaded onto the column without significantly increasing the volume. Collect both the positive and negative fractions*. Centrifuge the positive fraction at 300 x g for 3 min. Discard the supernatant and resuspend in 2 ml of RPMI + 5% FBS. Take 10 µl and add to 90 µl trypan blue before counting neutrophils on a hemocytometer.
    4. *The negative fraction is saved because it can be re-run if there is an issue with the machine and the neutrophils are not separated. It can be discarded once you have confirmed the presence of neutrophils in the positive fraction. Alternatively, the negative fraction can be used to generate bone marrow derived macrophages.
    Notes:
    1. Turks solution is used to count cells in collected bone marrow because it is effective at lysing red blood cells that are not lysed by the TAC treatment. It is important to lyse red blood cells because it makes it much easier to count white blood cells.
    2. The expected yield from tibiae and femurs from two BL/6J mice is around 1 x 108 cells. The expected yield of neutrophils from two BL/6J mice is usually at least 1.4 x 107cells.
    3. Neutrophil purification is monitored by light microscopy during counting on the hemocytometer. Additional confirmation can come from labeling with anti-Ly6G antibodies and analysis with flow cytometry (see [Swamydas and Lionakis, 2013]).
    4. The details of this process will vary depending on the specific magnetic cell sorter. These details are for the AutoMACS (Miltenyi), although purification can be performed manually with Miltenyi columns instead.
    5. A video explaining clearly how to use the AutoMACS is available from the manufacturer. See the following URL: https://www.youtube.com/watch?v=h06klEgji4o.
  4. Biotinylation and fluorescent strepavidin labeling of C. albicans hyphal cell wall.
    Note: To examine interaction of neutrophils with the cell wall, a fluorescent label can be used. This labeling can be done in parallel with the neutrophil separation from bone marrow. While we did not typically use this step in the experiments involved in looking at NETs, it was used extensively in other assays looking at neutrophil mediated changes to the fungal cell wall. A basic diagram of the C. albicans cell wall is shown in Figure 1A. Detailed schematics of C. albicans cell wall are widely available in the literature (see [Perez-Garcia et al., 2011]):  
    1. This protocol uses the FluoReporter® Cell-Surface Biotinylation Kit. In this kit, amine-reactive biotin bonds covalently with cell wall proteins. Using a new kit, take one biotin vial and resuspend it in 250 µl DMSO to make a 0.2 mg/ml stock solution of biotin-XX SSE. This can be stored at -20 °C and reused in later experiments until gone.
    2. With your C. albicans at 3 x 108 cells/ml, take enough volume so that you will have 3 x 107 cells for each sample you are going to run. To maintain this concentration, do all wash steps with the same volume of liquid and with 30 sec spins at 15,000 x g in a microcentrifuge.
    3. Wash once with PBS + Na2CO3 at pH 8 (see Recipes).
    4. Resuspend the hyphae in PBS + Na2CO3 (pH 8) and stain with biotin-XX SSE at 0.01 µg/µl for 15 min at room temperature.
    5. Wash three times with PBS pH 7.2. Resuspend in PBS pH 7.2.
    6. Stain with a fluorescently labeled streptavidin at 36 µg/ml for 30 min at room temperature.
    7. Wash once with PBS pH 7.2 and resuspend in PBS pH 7.2 until ready for use with neutrophils.
    Note: The fluorescent label used will depend on the experiment. During cell wall damage experiments, we typically used Alexa Fluor 647-conjugated streptavidin so that green and red fluorescent channels could be used for other labels.
  5. Hyphal/Immune cell interaction (2.5 h)
    Incubate 3 x 107 hyphal cells with or without 7 x 106 neutrophils in 1.7 ml microcentrifuge tubes with RPMI + 5% FBS, in a total of 1.7 ml, on a rotator at 37 °C for 2.5 h. The sample without neutrophils serves as a control.
    Note: It is important to have the microcentrifuge tube almost completely full of media so that neutrophils and hyphae do not get trapped and dried out on the sides of the tube in an air pocket. 
  6. Staining for NET visualization (3 h; for a schematic diagram of the staining process see Figure 3A)


    Figure 3. Schematic of the staining steps of the protocol

    1. Note that all steps are done on ice and with ice-cold buffers, unless specified otherwise. This prevents any additional metabolic functions in these live cells.
    2. Spin down the samples in a microcentrifuge at max speed (15,000-21,000 x g) for 30 sec. Remove the supernatants and wash once with 500 µl of PBS.
    3. Resuspend in 200 µl of PBS + 2% BSA to block the samples. Incubate at room temperature for 30 min.
    4. At this point, split all the samples in half, one will receive primary antibody and the other will not to serve as a control.
    5. Spin samples in the microcentrifuge tube and discard the supernatant.
    6. Resuspend the samples in 100 µl PBS + 2% BSA with primary antibody or with just PBS + 2% BSA (no primary control). The primary antibody can either be anti-myeloperoxidase (0.1 mg/ml) or anti-histone H3 citrulline R2+R8+R17 (0.014 mg/ml).
    7. Incubate the samples on ice for 1 h.
    8. Wash the samples 3-5 times with 500 µl PBS.
    9. After the final wash, resuspend all samples (both the primary and the no primary controls) in a total of 100 µl PBS + 2% BSA with Sytox Green (156 nM), calcofluor white (25 ng/ml) and secondary antibody. For the samples stained with anti-MPO primary, the secondary antibody we used was donkey anti-goat IgG Cy3 (0.007 mg/ml) and for the samples stained with anti-histone, the secondary we used was donkey anti-rabbit IgG Cy3 (0.0075 mg/ml).
      Note: Sytox Green is a non-membrane permeable DNA stain which will fluoresce brightly after binding the extracellular DNA found in NETs. CFW is a stain for chitin in the fungal cell wall.
    10. Incubate for 30 min at room temperature.
    11. After incubation, wash the samples 3-5 times with PBS and resuspend in 35 µl of PBS.
  7. Visualizing neutrophil mediated disruptions of the fungal cell wall (for a schematic diagram of the staining process see Figure 3B)
    1. As an alternative to staining for NET components, a slight alteration of the protocol will allow the visualization of neutrophil mediated changes to the fungal cell wall. This involves staining with sDectin-1-Fc to look for the exposure of the inflammatory fungal pathogen associated molecular pattern β-glucan, staining with calcofluor white to look for changes in fungal chitin and the fluorescent labeling of the outer cell wall as outlined in step 4 to examine immune mediated disruptions of this outer layer. Steps 1-5 are followed as outlined above but step 6 is skipped.
    2. Spin down the samples in a microcentrifuge at max speed for 30 sec. Remove the supernatants and wash once with 500 µl of PBS.
    3. Resuspend in 200 µl of PBS + 2% BSA to block the samples. Incubate at room temperature for 30 min.
    4. Resuspend the samples in a total of 50 µl PBS + 2% BSA with sDectin-1-Fc at 17 µg/ml. Incubate on ice for 1 h.
    5. Wash the samples 3-5 times with 500 µl PBS.
    6. After the final wash, resuspend all samples in a total of 200 µl of PBS + 2% BSA with calcofluor white (CFW; 25 ng/ml) and donkey anti-human IgG Cy3 secondary antibody (0.8 mg/ml). Incubate for 30 min at room temperature. After incubation, wash the samples 3-5 times with PBS and resuspend in 35 µl of PBS.
      Note: In order to reduce the amount of sDectin-1-Fc used per sample, the sDectin-1-Fc staining step is typically done in a smaller volume (50 µl) than the blocking or secondary antibody steps which are done with 200 µl.
    7. The samples are now ready to visualize by fluorescence microscopy. Using clean glass slides, load 5 µl of sample and place a coverslip on top. The coverslip can be sealed with nail polish to prevent drying out during imaging. Depending on the experimental question, random fields of view or specific sites were chosen and imaged.
    8. Once set, the exposure times for all channels were kept the same for all images. An equal number of images were obtained from all samples for use in analysis.
    9. The hyphae will have the chitin in their cell walls stained with the CFW. Areas of exposed β-glucan that is bound by sDectin-1-Fc will be marked by Cy3 fluorescent signal (shown via diagram in Figure 1B and via microscopy in Figure 4). Areas of neutrophil attack of the outer cell wall can be detected by examining the loss of fluorescent streptavidin signal (Figure 4A). A viability indicator is usually included by using a C. albicans strain with cytoplasmic fluorophore expression. For these experiments, a prototrophic WT-GFP strain (Genotype: SC5314 Peno1::Peno1-EGFP-NATR; [Wheeler et al., 2008]) was typically used.

Data analysis

  1. For NET staining, analysis focused on the qualitative evaluation of whether MPO and citrullinated H3 signals were present in the stained samples, co-localized with extracellular DNA and did not appear in the no primary controls (Figure 5).
  2. For analysis of cell wall damage, a number of options are available and depend on the hypothesis being tested. For images with z-stacks, we first create maximum image projections using the standard Zeiss Axiovision software (Zeiss Microscopy). One way we have analyzed these data is by examining the percentage of viable cells in all images which display specific cell wall changes (Figure 4D). This type of analysis is useful when attempting to determine if specific inhibitors or genetic knockouts, on either the fungal or neutrophil side, disrupt the process. We have also analyzed this data by determining the mean fluorescent intensity (MFI) of the staining at specific sites of interest and comparing this to the MFIs seen in controls (Figure 4C). The MFI can be obtained with numerous programs. We captured our images with a Zeiss Axiovision microscope and determined the MFI with Axiovision software. This analysis has proven useful in quantitatively measuring the overlap of specific cell wall changes (for example: increased sDectin-1-Fc staining overlaps at areas of increased CFW staining at neutrophil attack sites). It has also proven useful in demonstrating the changes in levels of cell wall components at specific sites when using fungal strains with fluorescent protein fusions.

Notes:

  1. In order to do these comparisons, all images being compared within an experiment (experimental samples and controls) must be acquired with the same exposure times. An equal number of images for all samples should also be obtained and analyzed. We usually use between 12 and 20 fields of view per sample per experiment. These fields of view can be obtained randomly or by finding specific sites depending on the experiment; note that fields should be chosen using a neutral channel (such as brightfield) to eliminate potential bias. Typically, three independent experiments were performed and data are shown as the average of the three experiments ± standard error. Note that here quantification of only a single field is shown so there are no error bars.
  2. When analyzing images, we avoid including the tips of hyphae in the analysis. These tips can be the sites of new growth that were not originally biotinylated and this growth can cause cell wall changes unrelated to the interaction with neutrophils that would complicate the analysis. Examples of these areas are shown in Figures 4E-4F. We also avoid hyphae that are overlapping each other, especially when examining MFI, to make sure each hyphal segment is well resolved.


    Figure 4. In vitro staining of hyphal cell wall after neutrophil attack. Representative results for the sDectin-1-Fc staining assay are shown for an attack site and for the no neutrophil control. Panel A illustrates typical staining of both a site attacked by a neutrophil (top) and by the normal unattacked cell wall in a sample of C. albicans hyphae not challenged with neutrophils (bottom). Panel B shows how fluorescence intensity of different labels is measured using a region-of-interest box in Axiovision software to obtain the mean fluorescence intensity (MFI) for each channel. The data comparing fluorescence intensity between a single attack site and a no neutrophil control site are shown in panel C. In addition to quantitative measurement of fluorescence intensities, the percentage of cells displaying a given phenotype (over a number of fields imaged) is also quantified. Panel D shows representative results from quantification of two single fields of view shown in panels E and F. Examples of hyphal tips, which were avoided during analysis, are indicated with white brackets (E-F). More extensive analysis can be seen in Hopke et al., 2016. Scale bar represents 10 µm (A) or 20 µm (E-F).


    Figure 5. NET staining assay. Representative results for the NET staining assay are shown for both MPO (A & inset) and citrullinated histone H3 (C & inset). The corresponding secondary antibody only controls are shown below each (B and D). Panels shown include the individual channels for Sytox green staining and either MPO or citrullinated H3 staining as well as the overlay of these channels. Scale bar represents 10 µm (insets) or 20 µm (A-B, C-D).
  1. Representative results
    Representative staining results for NET components are shown in Figure 5. C. albicans hyphae staining by CFW is shown in blue. Extracellular DNA staining by Sytox is shown in green. Staining with MPO (Figures 5A-5B) or citrullinated H3 histone antibody (Figures 5C-5D) is shown in red. The no primary (NP) controls show a lack of any signal in the red channel, demonstrating the specificity of the primary antibody during the staining process (Figures 5B and 5D). MPO staining signal co-localizes with extracellular DNA but also directly on hyphae, suggesting MPO is released by neutrophils during contact with hyphae both within and beyond the context of NETs (Figure 5A, inset). Citrullinated histone H3 signal localizes with extracellular DNA staining only, suggesting it is only released by neutrophils in the context of NETs and stays closely bound to DNA (Figure 5C, inset). Co-localization of staining with all three classical NET markers was used to demonstrate true NETs were being formed during incubation of neutrophils with hyphae.
    Representative results for staining to examine fungal cell wall damage are shown in Figure 4A neutrophil attack site is shown, with increased CFW staining, strong sDectin-1-Fc staining and a loss of Alexa Fluor 647-conjugated Streptavidin signal (Figure 4A). A site from the no neutrophil control sample is also shown for comparison. These controls show the phenotype of the normal hyphal cell wall. Examples of data analysis from experiments done with this protocol are also shown including the mean fluorescence intensity (MFI) for different cell wall stains at both the attack and control site (Figures 4B-4C) and the percentage of viable cells showing cell wall changes (Figure 4D) from example images (Figures 4E-4F).

Recipes

  1. PBS, pH 7.2
    154 mM sodium chloride
    5.6 mM sodium phosphate, dibasic anhydrous
    1.06 mM potassium phosphate, monobasic anhydrous
    Adjust pH to 7.2
    Autoclave for 20 min on liquids cycle
  2. PBS + Na2CO3, pH 8
    Prepare a 1 M solution of Na2CO3, pH 10.5
    Add 1/10 volume to PBS to increase pH to ~8 to enhance amine-reactive chemistry
  3. YPD broth
    20 g/L BD Bacto peptone
    10 g/L BD Bacto yeast extract
    20 g/L dextrose
    Fill to 1 L with distilled water
    Autoclave at 121 °C for 20 min on liquids cycle
    Note: YPD agar is the same as above with the addition of 20 g/L BD Bacto agar. After autoclaving, wait for the bottle to cool to the point it can be handled and make agar plates by adding 25 ml molten agar to 100 x 20 mm Petri dishes. Store at 4 °C until ready to use.
  4. PBS + 5% FBS
    47.5 ml PBS
    2.5 ml heat inactivated fetal bovine serum
    Note: We typically heat-inactivate FBS by placing a 500 ml bottle in a 55 °C water bath for 20 min. We then pre-aliquot the FBS by adding 2.5 ml to 50 ml conicals and freezing them at -20 °C. They can then be thawed when ready to use. Making up two tubes of this is usually enough for one experiment.
  5. PBS + 2% FBS
    24 ml PBS
    16 ml PBS + 5% FBS
    Note: You can make up the PBS + 2% FBS directly or you can dilute some of the PBS + 5% FBS that you will have already made as outlined above.
  6. PBS + 2% BSA
    50 ml PBS
    1 g Probumin® bovine serum albumin diagnostic grade powder
    Note: The PBS + 2% BSA is stored at 4 °C until just before use. Be sure to warm it up to room temperature before opening cap to prevent condensation of moisture inside bottle.
  7. RPMI + 5% FBS
    RPMI 1640, with 25 mM HEPES and L-glutamine
    Heat inactivated fetal bovine serum
    Note: This solution is made up fresh each day for the experiment. The amount needed will depend on the number of samples you are running that day.
  8. Tris-HCl, pH 8.0
    1 M Tris base
    Adjust pH to 8.0 with concentrated hydrochloric acid
  9. TAC buffer (3 ml for 1 min)
    0.017 M Tris-HCl, pH 8.0
    0.114 M NH4Cl
  10. Turks solution
    0.01% crystal violet
    3% acetic acid

Acknowledgments

This work has been supported by grants from the following sources: the U.S. Department of Agriculture (ME0-H-1-00517-13), the National Institutes of Health (R15AI094406), and the Burroughs Wellcome Fund to Dr. Robert T. Wheeler. This manuscript is Maine Agricultural and Forest Experiment Station Publication Number 3481.

References

  1. Amulic, B., Cazalet, C., Hayes, G.L., Metzler, K.D., Zychlinsky, A. (2012). Neutrophil function: from mechanisms to disease. Annu Rev Immunol (30): 459-489.
  2. Branzk, N., Lubojemska, A., Hardison, S. E., Wang, Q., Gutierrez, M. G., Brown, G. D. and Papayannopoulos, V. (2014). Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol 15(11): 1017-1025.
  3. Branzk, N. and Papayannopoulos, V. (2013). Molecular mechanisms regulating NETosis in infection and disease. Semin immunopathol (35): 513-530.
  4. Brown, G. D., Denning, D. W., Gow, N. A., Levitz, S. M., Netea, M. G. and White, T. C. (2012). Hidden killers: human fungal infections. Sci Transl Med 4(165): 165rv113.
  5. Bruns, S., Kniemeyer, O., Hasenberg, M., Aimanianda, V., Nietzsche, S., Thywissen, A., Jeron, A., Latge, J. P., Brakhage, A. A. and Gunzer, M. (2010). Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA. PLoS Pathog 6(4): e1000873.
  6. Graham, L. M., Tsoni, S. V., Willment, J. A., Williams, D. L., Taylor, P. R., Gordon, S., Dennehy, K. and Brown, G. D. (2006). Soluble Dectin-1 as a tool to detect beta-glucans. J Immunol Methods 314(1-2): 164-169.
  7. Hopke, A., Nicke, N., Hidu, E. E., Degani, G., Popolo, L. and Wheeler, R. T. (2016). Neutrophil attack triggers extracellular trap-dependent candida cell wall remodeling and altered immune recognition. PLoS Pathog 12(5): e1005644.
  8. Lionakis, M. S. and Netea, M. G. (2013). Candida and host determinants of susceptibility to invasive candidiasis. PLoS Pathog 9(1): e1003079.
  9. Perez-Garcia, L. A., Diaz-Jimenez, D. F., Lopez-Esparza, A. and Mora-Montes, H. M. (2011). Role of cell wall polysaccharides during recognition of Candida albicans by the innate immune system. Glycobiology 1.
  10. Rohm, M., Grimm, M. J., D’Auria, A. C., Almyroudis, N. G., Segal, B. H. and Urban, C. F. (2014). NADPH oxidase promotes neutrophil extracellular trap formation in pulmonary aspergillosis. Infect Immun 82(5): 1766-1777.
  11. Swamydas, M. and Lionakis, M. S. (2013). Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments. J Vis Exp (77): e50586.
  12. Urban, C. F., Reichard, U., Brinkmann, V. and Zychlinsky, A. (2006). Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol 8(4): 668-676.
  13. Wheeler, R. T., Kombe, D., Agarwala, S. D. and Fink, G. R. (2008). Dynamic, morphotype-specific Candida albicans beta-glucan exposure during infection and drug treatment. PLoS Pathog 4(12): e1000227.

简介

在本协议中,我们描述了如何在小鼠嗜中性粒细胞与真菌白色念珠菌的真菌菌丝共培养的背景下观察嗜中性粒细胞胞外捕获物(NETs)和真菌细胞壁变化。 这些方案很容易调整,以测试与免疫细胞对真菌和细胞壁的影响有关的各种假设,使其成为探索真菌感染期间宿主病原体相互作用的有前景的工具。

C。白色念珠菌是一种多态性机会性酵母,嗜中性粒细胞是免疫细胞,对于这种和其他真菌病原体的防御至关重要(Brown等,2012; Lionakis和Netea,2013)。 NETs是可以针对病原体部署的潜在防御机制,并且已经表明它们优先针对诸如C的微生物细胞部署。白念珠菌菌丝太大而不能吞噬(Urban et al。,2006; Bruns等人,2010; Branzk等人,,2014; Rohm等人,2014)。 NET已经显示含有许多组分,包括髓过氧化物酶,胞外DNA和瓜氨酸化的组蛋白(Amulic等人,2012; Branzk和Papayannopoulos,2013)。为了阳性鉴定NETs,体外实验和实验中的标准包括染色并显示这些标记物的共定位。虽然NETs对防御的确切贡献。白色念珠菌感染尚不清楚,我们的小组已经证明,他们可以在C引起应激反应和细胞壁重排。白色菌菌丝。具体来说,NET攻击导致更大的甲壳素沉积和β-葡聚糖暴露,如图1所示。这些多糖通常位于甘露聚糖层下面,并且它们的暴露可以改变免疫识别(Perez-Garcia et al。 ,,2011)。这里描述的基本测定被广泛用于我们的组(Hopke等人,2016)探测这个主题。虽然这里概述了检测NETs和真菌细胞壁变化的目的,该协议容易调整,以利用化学抑制剂,转基因或敲除真菌菌株或小鼠嗜中性粒细胞和其他染色目标的许多组合来测试各种各样的假设(Hopke 等人,2016)。因此,该方案代表了一种有希望的方法来进一步阐明免疫细胞对C的影响。白色念珠菌细胞壁,其应激反应以及改变的表位暴露于宿主防御真菌感染的重要性。


图1.细胞壁组织前和嗜中性粒细胞攻击示意图。在稳态条件下,菌丝细胞壁由三种主要多糖组分(甘露聚糖,β-葡聚糖和壳多糖)组成,但是大多数几丁质和β-葡聚糖由于位于甘露聚糖层之下而无法进行识别。攻击后,细胞壁甘露糖蛋白丧失和几丁质含量增加。这些变化导致β-葡聚糖的更大的表面识别,也可能是几丁质。

关键字:真菌, 白色念珠菌, NETs, 宿主 - 病原体相互作用, 细胞壁, sDectin-1-Fc

材料和试剂

  1. 防护手套和实验室外套
  2. 弗林特玻璃培养管16 x 150毫米(VWR,目录号:60825-435)
  3. 50ml锥形管(Corning,Falcon ®,目录号:352098)
  4. Fisherbrand 100 x 20毫米培养皿(Fisher Scientific,目录号:FB0875711Z)
  5. 10ml注射器(BD,目录号:301604)
  6. 25 G 5/8针(BD,目录号:305122)
  7. 70微米的细胞过滤器(Corning,Falcon ®,目录号:352350)
  8. 1.7ml微量离心管
  9. 普通显微镜幻灯片(VWR,目录号:48300-025)
  10. Kim-Kap TM 一次性闭合; 16毫米(Kimble Chase生命科学研究产品,目录号:7366316)
  11. Miltenyi列(Miltenyi Biotec,目录号:130-021-101)
  12. WT-FarRed670菌株。白色念珠菌(Hopke等人,2016)
  13. WT-GFP株(Wheeler等人,2008)
  14. C57BL/6J小鼠,6-10周龄(JACKSON LABORATORIES,目录号:000664)
  15. 甘油(EMD Millipore,目录号:GX0185-5)
  16. 抗Ly6G生物素抗体(Thermo Fisher Scientific,eBioscience,目录号:13-5931-85)
  17. 抗生物素磁珠(Miltenyi Biotec,目录号:130-090-485)
  18. 台盼蓝(Lonza,目录号:17-942E)
  19. FluoReporter ®细胞表面生物素化试剂盒(Thermo Fisher Scientific,Molecular Probes TM,目录号:F20650)
  20. Alexa Fluor 647缀合的链霉亲和素(Jackson ImmunoResearch,目录号:016-600-084)
  21. 抗髓过氧化物酶(MPO)(R& D Systems,目录号:AF3667)
  22. 抗组蛋白H3(瓜氨酸R2 + R8 + R17)(Abcam,目录号:ab5103)
  23. Sytox Green(Thermo Fisher Scientific,Molecular Probes TM ,目录号:S7020)
  24. Calcofluor白/荧光增白剂28(Sigma-Aldrich,目录号:F3543)
  25. 驴抗山羊IgG Cy3(Jackson ImmunoResearch,目录号:705-165-147)
  26. 驴抗兔IgG Cy3(Jackson ImmunoResearch,目录号:711-165-152)
  27. sDectin-1-Fc(根据[Graham等人,2006])在家制造)
  28. 驴抗人IgG Cy3(Jackson ImmunoResearch,目录号:709-165-149)
  29. 指甲油
  30. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-3)
  31. 无水磷酸氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:S374-500)
  32. 磷酸氢钾,一水合无水(KH 2 PO 4)(Fisher Scientific,目录号:P285-500)
  33. 碳酸钠(Na 2 CO 3)(Sigma-Aldrich,目录号:S7795)
  34. BD Bacto蛋白胨(BD,Bacto TM,目录号:211677)
  35. BD Bacto酵母提取物(BD,Bacto TM,目录号:212750)
  36. 葡萄糖(Fisher Scientific,目录号:D-163)
  37. BD Bacto琼脂(BD,Bacto TM,目录号:214014)
  38. 加热灭活的胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:10082147)
  39. Probumin ®牛血清白蛋白诊断级粉末(EMD Millipore,目录号:820451)
  40. RPMI,25 mM HEPES和L-谷氨酰胺(Lonza,目录号:12-115F)
  41. 氯化铵(NH 4 Cl)(EMD Millipore,目录号:AX12701)
  42. 结晶紫(Sigma-Aldrich,目录号:C0775)
  43. 乙酸(Fisher Scientific,目录号:A38-212)
  44. Tris碱(AMRESCO,目录号:0497)
  45. 盐酸(HCl)(Fisher Scientific,目录号:A144-500)
  46. PBS,pH 7.2(参见食谱)
  47. PBS + Na 2 CO 3,pH 8(参见食谱)
  48. YPD肉汤(见食谱)
  49. PBS + 5%FBS(见食谱)
  50. PBS + 2%FBS(参见食谱)
  51. PBS + 2%BSA(参见食谱)
  52. RPMI + 5%FBS(见配方)
  53. Tris-HCl,pH 8.0(参见食谱)
  54. TAC缓冲(见配方)
  55. 土耳其人解决方案(见配方)

设备

  1. 滚筒(Eppendorf,新不伦瑞克科学)和孵化器(VWR)
  2. 血细胞计数器用于细胞计数(Hausser Scientific,目录号:1492)
  3. 分光光度计(Eppendorf; Biophotometer)
  4. 用适用于15和50毫升锥形管的离心机(Thermo Fisher Scientific,型号:Sorvall TM Legend TM RT)
  5. AutoMACs分离系统(Miltenyi Biotec,型号:autoMACS ®//Pro Pro Separator)
  6. 蔡司Axiovision荧光显微镜(Zeiss;定制)
  7. 解剖套件(VWR,目录号:631-0616)
  8. pH计(Fisher Scientific,型号:accumet TM Excel XL15)
  9. 用于1.7ml管的微量离心机(Eppendorf,型号:5424)
  10. 高压灭菌器
  11. 500毫升瓶(VWR,目录号:89000-233)
  12. 水浴

软件

  1. AxioVision Rel48软件(Zeiss: https://www.zeiss.com/microscopy/us/downloads/axiovision-downloads.html
  2. Photoshop(Adobe Systems,San Jose,CA)

程序

  1. 白念珠菌菌丝生长(3天;参见图2A的步骤示意图)

    图2.协议前三个步骤的原理图

    1. 条纹适当的菌株C。白色念珠菌与来自冷冻的25%甘油原液的单个菌落在YPD琼脂上(参见食谱)并在37℃下孵育过夜。对于这些实验,我们使用WT-FarRed670菌株。白色念珠菌(基因型:P ENO1 :: P ENO1 -FarRed670-NAT R ; [Hopke等人,2016])。
    2. 第二天挑一个殖民地并转移到5ml的YPD液体中(见食谱)。我们将5毫升的16毫升150毫米火石玻璃管放上盖子,并在30℃下在滚筒上孵育过夜。收获YPD培养物并使用血细胞计数器计数酵母。
    3. 以2.5×10 6细胞/ml的浓度稀释到30ml RPMI中,并分成5ml等分试样。在滚筒上在30℃下孵育过夜。将RPMI培养液倒入50ml锥形管中,以3,000×g离心5分钟。去除大部分上清液(因此只剩下约5毫升的残留物),然后轻轻地将沉淀物重新悬浮在剩余的液体中。由于菌丝的形态,您不能使用血细胞计数器来确定细胞数量,而是使用分光光度计来估计菌丝的浓度。 600的OD 600相当于1×10 7个细胞/ml。虽然通过OD 600定量菌丝不如量化酵母那样准确,但是我们发现它提供了可重复的结果。调整至3×10 8细胞/ml以用作测定中的储备溶液。
      注意:您将在小体积内具有高度集中的菌丝数量,因此最好在找到OD <600> 之前进行稀释,以确保您落在可读范围内并保存细胞。我们的比色皿通常需要1毫升,所以我们取100微升菌丝并加入到900微升PBS中。 PBS将作为您的空白对照。
  2. 分离小鼠骨髓(1-2小时;参见图2B的程序原理图;详细视频方案参见[Swamydas和Lionakis,2013])
    1. 通过CO 2吸入安慰两个6-10周龄的两只C57BL/6J雌性小鼠,然后颈椎脱位。解剖后腿获取股骨和胫骨。取出所有的肉,并用PBS + 5%FBS转移到培养皿中(见食谱)。 
    2. 用PBS + 5%FBS填充10ml注射器,并附上25 G 5/8针。将针插入骨头并排出注射器中的液体以将骨髓推出培养皿(如果难以插入针头,则可以夹住骨骼的两端)。
    3. 当所有骨髓已经释放到培养皿中时,将其通过70μm的细胞过滤器并转移到50ml圆锥形中。以300×g离心3分钟。
    注意:
    1. 最好用没有软组织残留的培养皿驱除骨髓。组织会堵塞针头。如果需要,您可以在一个培养皿中完成骨骼清洁,然后将骨骼移至另一个PBS + 5%FBS,然后驱除骨髓。
    2. 所有动物研究均按照"国家卫生研究院实验动物护理和使用指南"中的建议进行。动物通过吸入二氧化碳来安乐死。感染动物每天监测两次感染迹象,并对病态动物进行安乐死。 UMaine IACUC /伦理委员会批准了该协议
  3. 通过AutoMACs从小鼠骨髓中分离嗜中性粒细胞(1.5-2小时;参见图2C的程序示意图)
    1. 弃去上清液,并用圆锥形管重新悬浮沉淀。加入3ml Tris /氯化铵(TAC)缓冲液(见Recipes),并在室温下孵育1分钟以溶解任何红细胞。孵育后,用PBS + 2%FBS稀释至30ml。取10μl骨髓溶液,加入40μl土耳其人溶液(参见食谱)。在血细胞计数器上计数细胞。
    2. 将骨髓溶液以300 x g离心3分钟。丢弃上清液并轻轻将沉淀重悬于PBS + 2%FBS中,浓度为1×10 8个细胞/ml。与抗-IL6G生物素抗体(0.5mg/ml的储备)一起在1μl抗体/100μl骨髓中在冰上孵育15分钟。通过在300×g离心3分钟并且弃去上清液洗涤。重悬于10ml PBS + 5%FBS中,然后再次离心。弃去上清液,再次悬浮于10ml PBS + 5%FBS中。与抗生物素磁珠(10μl/1×10 7个细胞)一起在冰上孵育30分钟。在整个孵化期间定期搅动。如上所述用10ml PBS + 5%FBS洗涤一次。将沉淀重悬于1ml PBS + 5%FBS中 注意:在此期间,打开AutoMACS并运行CLEAN循环。
    3. 一旦完成,将正面和负面的口中清洁出50ml锥形管。将骨髓放在进气口并运行正选择程序。当除了0.2毫升体积以外的所有摄入量中,加入1毫升PBS + 2%FBS。这确保了所有的残留样品都被加载到色谱柱上,而不会显着增加体积。收集正负分数*。以300×g离心阳性部分3分钟。弃去上清液并重悬于2ml RPMI + 5%FBS中。在血细胞计数器上计数嗜中性粒细胞前,取10μl加入90μl台盼蓝。
    4. *负数部分被保存,因为如果机器出现问题并且嗜中性粒细胞不分离,则可以重新运行。一旦您确认阳性部分中存在嗜中性粒细胞,就可以将其丢弃。或者,阴性部分可用于产生骨髓来源的巨噬细胞。
    注意:
    1. 土耳其人溶液用于对收集的骨髓中的细胞进行计数,因为它有效地裂解不经TAC治疗裂解的红细胞。裂解红细胞是重要的,因为它可以更容易地计数白细胞。
    2. 来自两只BL/6J小鼠的胫骨和股骨的预期产量约为1×10 8个细胞。来自两只BL/6J小鼠的嗜中性粒细胞的预期产量通常至少为1.4×10 7个细胞。
    3. 在计数血细胞计数器时,通过光学显微镜监测嗜中性粒细胞纯化。额外的确认可来自用抗Ly6G抗体标记并用流式细胞仪分析(参见[Swamydas和Lionakis,2013])。
    4. 该过程的细节将根据特定的磁性细胞分选器而变化。这些细节适用于AutoMACS(Miltenyi),尽管可以使用Miltenyi列手动进行纯化。
    5. 可以从制造商处获得一个解释如何使用AutoMACS的视频。请参阅以下网址: https://www.youtube.com/watch?v=h06klEgji4o
  4. 生物素化和荧光链霉亲和素标记。白色念珠菌细胞壁。
    注意:为了检查嗜中性粒细胞与细胞壁的相互作用,可以使用荧光标记。该标记可以与从骨髓分离的嗜中性粒细胞平行进行。虽然我们通常不会在参与NETs的实验中使用这一步骤,但是在其他测定中广泛使用这一步骤,可以看到嗜中性粒细胞介导的真菌细胞壁的变化。白色念珠菌细胞壁的基本图示于图1A中。白色念珠菌细胞壁的详细原理图在文献中广泛可用(参见[Perez-Garcia et al。,2011]):  
    1. 该方案使用FluoReporter ®细胞表面生物素化试剂盒。在该试剂盒中,胺反应性生物素与细胞壁蛋白共价键合。使用一个新的试剂盒,取一个生物素小瓶,并将其重新悬浮于250μlDMSO中以制备0.2mg/ml生物素-XX SSE的储备溶液。这可以保存在-20°C,并在以后的实验中重新使用,直到不见了。
    2. 与您的 C。白细胞在3×10 8细胞/ml,取足够的体积,以便您将要运行的每个样品具有3×10 7 细胞。为了保持这个浓度,在微量离心机中用相同体积的液体进行所有的洗涤步骤,并以15,000 x g的速度旋转30秒。
    3. 在pH8下用PBS + Na 2 CO 3洗涤一次(参见食谱)。
    4. 将菌丝重悬于PBS + Na 2 CO 3(pH 8)中,并在室温下以0.01μg/μl的生物素-XO SSE染色15分钟。 >
    5. 用PBS pH 7.2洗三次。重悬于PBS pH 7.2。
    6. 用36μg/ml荧光标记的链霉亲和素在室温下染色30分钟。
    7. 用PBS pH 7.2洗涤一次,并重悬于PBS pH 7.2中直至准备好与嗜中性粒细胞一起使用。
    注意:使用的荧光标记将取决于实验。在细胞壁破坏实验期间,我们通常使用Alexa Fluor 647缀合的链霉抗生物素蛋白,以便绿色和红色荧光通道可用于其他标签。
  5. 呼吸/免疫细胞相互作用(2.5小时)
    在具有RPMI + 5%FBS的1.7ml微量离心管中,将总共1.7ml的3×10 7细胞细胞与有或没有7×10 6个嗜中性粒细胞孵育,总共1.7ml旋转器在37℃下搅拌2.5小时。不含嗜中性粒细胞的样品作为对照 注意:重要的是让微量离心管几乎完全充满介质,以使嗜中性粒细胞和菌丝不会被困在气管两侧的管中并被干燥。 
  6. NET可视化染色(3 h;染色过程示意图见图3A)


    图3.协议染色步骤示意图

    1. 注意,除非另有说明,否则所有步骤均在冰上和冰冷的缓冲液中进行。这可以防止这些活细胞中的任何其他代谢功能。
    2. 将微量离心机中的样品以最大速度(15,000-21,000 x g )旋转30秒。取出上清液,用500μlPBS洗一次。
    3. 重悬于200μlPBS + 2%BSA中以封闭样品。在室温下孵育30分钟。
    4. 在这一点上,将所有样本分成两半,一个将接收一次抗体,另一个将不作为对照。
    5. 在微量离心管中旋转样品,弃去上清液。
    6. 将样品重悬于含有一抗或100%PBS + 2%BSA(无主要对照)的100μlPBS + 2%BSA中。第一抗体可以是抗髓过氧化物酶(0.1mg/ml)或抗组蛋白H3瓜氨酸R2 + R8 + R17(0.014mg/ml)。
    7. 将样品在冰上孵育1小时。
    8. 用500μlPBS洗涤样品3-5次。
    9. 在最终洗涤后,将总共100μlPBS + 2%BSA的Sytox Green(156nM),calcofluor white(25ng/ml)和第二抗体重新悬浮所有样品(无论是初级对照组和无主要对照组)。对于用抗MPO初级染色的样品,我们使用的二抗是驴抗山羊IgG Cy3(0.007mg/ml),对于用抗组蛋白染色的样品,我们使用的是驴抗兔IgG Cy3(0.0075 mg/ml)。
      注意:Sytox Green是一种非膜可渗透的DNA染色剂,在结合NETs中发现的细胞外DNA后会发出明亮的荧光。 CFW是真菌细胞壁中几丁质的污渍。
    10. 在室温下孵育30分钟。
    11. 孵育后,用PBS洗涤样品3-5次,并重悬于35μlPBS中。
  7. 可视化嗜中性粒细胞介导的真菌细胞壁的破坏(染色过程的示意图参见图3B)
    1. 作为NET组分染色的替代方案,方案的轻微改变将允许中性粒细胞介导的真菌细胞壁变化的可视化。这涉及用sDectin-1-Fc染色以寻找炎症真菌病原体相关分子模式β-葡聚糖的暴露,用calcofluor白色染色以寻找真菌甲壳素的变化和外部细胞壁的荧光标记,如步骤4检查这种外层的免疫介导的破坏。如上所述遵循步骤1-5,但跳过步骤6。
    2. 将微量离心机中的样品以最大速度旋转30秒。取出上清液,用500μlPBS洗一次
    3. 重悬于200μlPBS + 2%BSA中以封闭样品。在室温下孵育30分钟。
    4. 将样品重悬在总共50μlPBS + 2%BSA中,以17μg/ml的sDectin-1-Fc。在冰上孵育1小时。
    5. 用500μlPBS洗涤样品3-5次。
    6. 最终洗涤后,将总共200μlPBS + 2%BSA与calcofluor白色(CFW; 25ng/ml)和驴抗人IgG Cy3二抗(0.8mg/ml)重新悬浮所有样品。在室温下孵育30分钟。孵育后,用PBS洗涤样品3-5次,并重悬于35μlPBS中。
      注意:为了减少每个样品使用的sDectin-1-Fc的量,sDectin-1-Fc染色步骤通常以比完成的阻断或二级抗体步骤更小的体积(50μl)进行用200μl。
    7. 样品现在可以通过荧光显微镜观察。使用干净的玻璃片,加载5μl样品,并在上面放置盖玻片。盖玻片可以用指甲油密封,以防止成像期间发生干燥。根据实验问题,选择随机视场或特定位点进行成像。
    8. 一旦设置,所有图像的所有通道的曝光时间保持不变。从所有样品中获得相等数量的图像用于分析。
    9. 菌丝将其细胞壁中的几丁质染色为CFW。由sDectin-1-Fc结合的暴露的β-葡聚糖的区域将被Cy3荧光信号(通过图1B中的图示和通过图4中的显微镜显示)标记。可以通过检查荧光抗生蛋白链菌素信号的损失来检测外细胞壁的嗜中性粒细胞攻击区域(图4A)。通常使用"C"可以包括生存力指标。白念珠菌菌株与细胞质荧光团表达。对于这些实验,可以使用营养型WT-GFP(Genotype:SC5314 Peno1 :: Peno1-EGFP -NAT),[Wheeler等人]。 ,2008])。

数据分析

  1. 对于NET染色,分析集中在定性评估MPO和瓜氨酸H3信号是否存在于染色样品中,与细胞外DNA共定位,并没有出现在无主要对照(图5)中。
  2. 对于细胞壁损伤的分析,有许多选项可用,并且取决于被测试的假设。对于具有z-stack的图像,我们首先使用标准的Zeiss Axiovision软件(Zeiss Microscopy)创建最大图像投影。我们分析了这些数据的一种方法是检查显示特定细胞壁变化的所有图像中活细胞的百分比(图4D)。当尝试确定特异性抑制剂或真菌或嗜中性粒细胞一侧的遗传敲除是否会破坏该过程时,这种分析是有用的。我们还通过确定感兴趣的特定位点的染色的平均荧光强度(MFI)并将其与对照中看到的MFI(图4C)进行比较,分析了该数据。 MFI可以通过许多程序获得。我们用Zeiss Axiovision显微镜捕获了我们的图像,并用Axiovision软件确定了MFI。该分析已被证明可用于定量测量特异性细胞壁变化的重叠(例如:在嗜中性粒细胞攻击部位增加的CFW染色区域增加的sDectin-1-Fc染色重叠)。当使用具有荧光蛋白融合的真菌菌株时,也证明可用于证明特定部位细胞壁组分水平的变化。

注意:

  1. 为了进行这些比较,在实验(实验样品和对照)中进行比较的所有图像必须以相同的曝光时间获得。还应获得并分析所有样品的相等数量的图像。我们通常每个实验使用12到20个视野。这些视野可以随机获得,也可以通过根据实验找到特定的部位;请注意,应使用中性通道(如明场)选择场,以消除潜在的偏差。通常,进行三次独立实验,数据显示为三次实验的平均值±标准误差。请注意,这里仅显示一个字段的量化,因此没有错误栏。
  2. 在分析图像时,我们避免在分析中包含菌丝的提示。这些提示可能是最初未生物素化的新生长的部位,这种增长可能导致与嗜中性粒细胞的相互作用无关的细胞壁变化,这将使分析复杂化。这些区域的示例在图4E-4F中示出。我们也避免了互相重叠的菌丝,特别是在检查MFI时,确保每个菌丝段得到很好的解决。


    图4.在中性粒细胞发作后菌丝细胞壁的体外染色针对攻击部位显示sDectin-1-Fc染色测定的代表性结果,对于无嗜中性粒细胞控制。图A示出了嗜中性粒细胞(顶部)和正常未被攻击的细胞壁在C样品中攻击的位点的典型染色。白色念珠菌菌丝未被嗜中性粒细胞(底部)挑战。图B显示如何使用Axiovision软件中的感兴趣区域测量不同标签的荧光强度,以获得每个通道的平均荧光强度(MFI)。单个攻击部位和无嗜中性粒细胞对照部位之间的荧光强度比较的数据显示在图C中。除荧光强度的定量测量之外,显示给定表型的细胞的百分比(在成像的多个场上)也被定量。图D显示了E和F图中所示的两个单一视场的定量的代表性结果。在分析期间避免的菌丝末端的实例用白色括号(E-F)表示。更广泛的分析可以在Hopke等人,2016中看到。比例尺表示10μm(A)或20μm(E-F)。


    图5. NET染色测定。针对MPO(A和插图)和瓜氨酸化组蛋白H3(C&插图)显示了NET染色测定的代表性结果。相应的仅次级抗体对照如下所示(B和D)。显示的面板包括用于Sytox绿色染色和MPO或瓜氨酸化H3染色的各个通道以及这些通道的重叠。刻度棒表示10μm(插图)或20μm(A-B,C-D)。
  1. 代表性成绩
    NET组件的代表性染色结果如图5所示。白色念珠菌染色由CFW显示为蓝色。 Sytox的细胞外DNA染色显示为绿色。用MPO染色(图5A-5B)或瓜氨酸化的H3组蛋白抗体(图5C-5D)显示为红色。无原代(NP)对照显示红色通道中没有任何信号,表明染色过程中第一抗体的特异性(图5B和5D)。 MPO染色信号与细胞外DNA共同定位,但也直接在菌丝上,表明在NETs内部和外部与菌丝接触期间,MPO被嗜中性粒细胞释放(图5A,插图)。瓜氨酸组蛋白H3信号仅定位于细胞外DNA染色,表明它仅在NETs的上下文中被嗜中性粒细胞释放并与DNA紧密结合(图5C,插图)。使用所有三种经典NET标记物的染色共定位来证实在嗜中性粒细胞与菌丝孵育期间正在形成真正的NETs。
    染色以检查真菌细胞壁损伤的代表性结果显示在图4A中,显示嗜中性粒细胞发生部位,增加CFW染色,强sDectin-1-Fc染色和Alexa Fluor 647缀合的链霉亲和素信号的丢失(图4A)。还显示了来自无嗜中性粒细胞对照样品的位点用于比较。这些对照显示正常菌丝细胞壁的表型。还显示了使用该方案进行的实验的数据分析的实例,包括在攻击和对照部位(图4B-4C)的不同细胞壁染色体的平均荧光强度(MFI)和显示细胞壁变化的活细胞的百分比图4D)从示例图像(图4E-4F)

食谱

  1. PBS,pH 7.2
    154 mM氯化钠
    5.6 mM磷酸钠,无水二碱性
    1.06mM磷酸钾,一水无水
    将pH调节至7.2
    高压灭菌20分钟液体循环
  2. PBS + Na 2 CO 3,pH 8
    制备1M的Na 2 CO 3,pH 10.5的溶液
    向PBS中加入1/10体积以将pH增加至〜8以增强胺反应性化学物质
  3. YPD肉汤
    20 g/L BD Bacto蛋白胨
    10g/L BD Bacto酵母提取物
    20 g/L葡萄糖
    用蒸馏水填充1L 液体循环时,在121°C高压灭菌20分钟 注意:YPD琼脂与上述相同,加入20g/L BD Bacto琼脂。高压灭菌后,等待瓶子冷却至可以处理的位置,并通过向100 x 20 mm培养皿中加入25 ml熔融琼脂制成琼脂平板。储存于4°C直到准备使用。
  4. PBS + 5%FBS
    47.5毫升PBS
    2.5 ml热灭活胎牛血清 注意:我们通常将500毫升瓶子放置在55°C水浴中20分钟,对FBS进行热灭活。然后,我们通过加入2.5毫升至50ml锥形物将FBS预分装,并将其在-20℃下冷冻。然后可以在准备使用时将其解冻。这样做的两个管通常足够用于一个实验。
  5. PBS + 2%FBS
    24毫升PBS
    16毫升PBS + 5%FBS
    注意:您可以直接补充PBS + 2%FBS,也可以稀释一些您将按上述方法制作的PBS + 5%FBS。
  6. PBS + 2%BSA
    50ml PBS
    1克Probumin ®牛血清白蛋白诊断级粉末
    注意:PBS + 2%BSA储存在4°C直到使用前。确保在打开盖子之前将其升温至室温,以防止瓶内水分冷凝。
  7. RPMI + 5%FBS
    RPMI 1640,含有25 mM HEPES和L-谷氨酰胺 热灭活的胎牛血清
    注意:这个解决方案每天都是新鲜的实验。所需金额将取决于当天运行的样本数量。
  8. Tris-HCl,pH 8.0
    1 M Tris碱基
    用浓盐酸调节pH至8.0
  9. TAC缓冲液(3ml,1分钟)
    0.017M Tris-HCl,pH8.0
    0.114 M NH 4 Cl
  10. 土耳其人解决方案
    0.01%结晶紫色
    3%乙酸

致谢

这项工作得到以下来源的资助:美国农业部(ME0-H-1-00517-13),美国国家卫生研究院(R15AI094406)和Burroughs Wellcome基金会Robert T. Wheeler博士。这份手稿是缅因州农林实验站出版号3481。

参考文献

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  2. Branzk,N.,Lubojemska,A.,Hardison,SE,Wang,Q.,Gutierrez,MG,Brown,GD and Papayannopoulos,V.(2014)。  中性粒细胞有意义的微生物大小,并响应大病原体选择性释放嗜中性粒细胞胞外捕获物。 em> 15(11):1017-1025。
  3. Branzk,N.和Papayannopoulos,V.(2013)。  调控感染和疾病中的NETosis的分子机制。 Semin immunopathol (35):513-530。
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  5. Bruns,S.,Kniemeyer,O.,Hasenberg,M.,Aimanianda,V.,Nietzsche,S.,Thywissen,A.,Jeron,A.,Latge,JP,Brakhage,AA和Gunzer,M。(2010) 。产生针对烟曲霉的细胞外捕获物体外和感染的肺组织依赖入侵的嗜中性粒细胞并受疏水蛋白RodA的影响。 PLoS Pathog 6(4):e1000873。
  6. Graham,LM,Tsoni,SV,Willment,JA,Williams,DL,Taylor,PR,Gordon,S.,Dennehy,K.and Brown,GD(2006)。< a class ="ke-insertfile"href = "http://www.ncbi.nlm.nih.gov/pubmed/16844139"target ="_ blank">可溶性Dectin-1作为检测β-葡聚糖的工具。 > 314(1-2):164-169。
  7. Hopke,A.,Nicke,N.,Hidu,EE,Degani,G.,Popolo,L。和Wheeler,RT(2016)。< a class ="ke-insertfile"href ="http: .ncbi.nlm.nih.gov/pubmed/27223610"target ="_ blank">嗜中性粒细胞攻击引发细胞外陷阱依赖性念珠菌细胞壁重塑和改变的免疫识别。 PLoS Pathog 12( 5):e1005644。
  8. Lionakis,MS和Netea,MG(2013)。 < em> Candida 和宿主对入侵性念珠菌病易感性的决定因素。 PLoS Pathog 9(1):e1003079。
  9. Perez-Garcia,LA,Diaz-Jimenez,DF,Lopez-Esparza,A.和Mora-Montes,HM(2011)。细胞壁多糖在天然免疫系统识别白色念珠菌期间的作用糖生物学 1.
  10. Rohm,M.,Grimm,MJ,D'Auria,AC,Almyroudis,NG,Segal,BH and Urban,CF(2014)。  NADPH氧化酶促进肺曲霉病中的嗜中性粒细胞胞外陷阱形成。 82(5):1766-1777 。
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  13. Wheeler,RT,Kombe,D.,Agarwala,SD and Fink,GR(2008)。  感染和药物治疗期间β-葡聚糖暴露的动态,形态特异性白色念珠菌。 4(12): e1000227。
  • English
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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. Hopke, A. and Wheeler, R. T. (2017). In vitro Detection of Neutrophil Traps and Post-attack Cell Wall Changes in Candida Hyphae. Bio-protocol 7(7): e2213. DOI: 10.21769/BioProtoc.2213.
  2. Hopke, A., Nicke, N., Hidu, E. E., Degani, G., Popolo, L. and Wheeler, R. T. (2016). Neutrophil attack triggers extracellular trap-dependent candida cell wall remodeling and altered immune recognition. PLoS Pathog 12(5): e1005644.
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