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Nov 2019

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Neutrophil Extracellular Trap Killing Assay of Candida albicans
白念珠菌中性粒细胞胞外诱杀试验   

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Abstract

Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized C. albicans; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized C. albicans. Neutrophil killing of opsonized C. albicans requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of C. albicans requires a washing step after allowing neutrophils to phagocytose the organism. By definition, NET kills organisms extracellularly. Therefore, it is important to skip the washing step and add an optimal ratio of neutrophils and C. albicans to the wells. To demonstrate the effect of NET, it is necessary to compare killing ability of neutrophils treated with micrococcal nuclease (MNase), an enzyme that digests NET, to that treated with heat-inactivated MNase. MNase is also applied to release NET-bound fungal elements for counting. This protocol can be applied to assay NET killing of other biofilm-forming organisms.

Keywords: Neutrophil extracellular trap (中性粒细胞胞外诱捕), Candida albicans (白色念珠菌), Neutrophil (中性粒细胞), Fungicidal activity (抑菌活性), Micrococcal nuclease (微球菌核酸酶)

Background

Candida albicans is an opportunistic fungal pathogen that resides as a commensal on mucosal surface and the skin in most humans. Environmental changes in temperature, nutrition, or the presence of serum induces its transformation from yeast form to hyphae. Candida infection is one of the top leading causes of overall healthcare-associated bloodstream infections in medical centers as well as regional hospitals. Mortality among patients with invasive candidiasis is as high as 40% even after receiving antifungal therapy (Brown et al., 2012; Chen et al., 2014; Kullberg and Arendrup, 2015). Patients with neutropenia and neutrophil dysfunction are at high risk for invasive candidiasis, suggesting the importance of neutrophil anti-Candida functions in host defense (Antachopoulos et al., 2007; Horn et al., 2009; Lionakis and Netea, 2013). Our work showed that mouse neutrophils utilize two independent mechanisms, one NADPH oxidase-dependent and the other NADPH oxidase-independent, to kill C. albicans. NADPH oxidase-dependent killing of opsonized C. albicans requires phagocytosis mediated by CR3, and NADPH oxidase-independent killing of unopsonized C albicans is through NET formation mediated by dectin-2 (Wu et al., 2017 and 2019). MNase is an enzyme that digests NET. To demonstrate the effect of NET, it is necessary to compare the killing ability of neutrophils that is treated with MNase to that treated with heat-inactivated MNase. Difference between the two treatments denotes killing by NET and not by other factors (Wu et al., 2019). Neutrophils are phagocytic. Taking up microorganisms through receptors triggers robust ROS production that kills the engulfed organism. To assay for NADPH oxidase-dependent killing of opsonized C. albicans, one of the important steps is to wash off un-engulfed microorganisms after allowing time for phagocytosis to take place (Vonk et al., 2012; Wu et al., 2017). In contrast, since NET kills microorganisms extracellularly, it is critical not to wash off un-engulfed organisms to assay for NET-mediated killing of C. albicans. Moreover, instead of lysing neutrophils by ddH2O at high pH (pH 11.0) to release ingested microorganisms, NET-forming neutrophils and fungal hyphal elements are detached from the wells by mini cell scraper and DNA digesting enzyme MNase (Wu et al., 2017 and 2019). The method described here were published in 2019 (Wu et al., 2019) to observe killing of C. albicans by NET. This method can be adapted to quantify NET-mediated killing of other biofilm-forming organisms.

Materials and Reagents

  1. Pipette tips
  2.  Mini cell scraper (Biotium, catalog number: 22003 )
  3.  Flat-bottomed 96-well plates (Corning, catalog number: 3599 )
  4. 15 ml conical tube (Corning, catalog number: 430791 )
  5. 90 mm x 15 mm Petri dish (Alpha Plus, catalog number: 16001 )
  6.  1.5 ml microcentrifuge tube (Corning, catalog number: MCT-150-C )
  7. 3 ml syringe with 23-gauge needle (BD, catalog number: 302111 )
  8. Parafilm (Bemis, catalog number: PM996 )
  9. C. albicans SC5314 strain (ATCC MYA-2876)
  10. Female inbred mice in C57BL/6JNarl background, 6-12 weeks of age (National Laboratory Animal Center, RMRC11005)
  11. Percoll (GE Healthcare, catalog number: 17-0891-01 )
  12. 10x Dulbecco's Phosphate Buffered Saline (DPBS) (Biological Industries, catalog number: 02-023-1A )
  13. 10x Hank's Balanced Salt Solution (HBSS) (Biological Industries, catalog number: 02-015-5A )
  14. 0.4% Trypan blue (Sigma-Aldrich, catalog number: 93595 )
  15. 7.5% sodium bicarbonate (Biological Industries, catalog number: 03-040-1B )
  16. Yeast-peptone-dextrose (YPD) broth (Bioshop, catalog number: YPD002.500 )
  17. Agar A (BIO BASIC, catalog number: FB0010 )
  18. 2 x 106 U/ml Micrococcal nuclease (MNase) (NEB, catalog number: M0247S )
  19.  Sterile double distilled H2O (ddH2O), adjusted to pH 11 by NaOH
  20. 1x Dulbecco's Phosphate Buffered Saline (DPBS) (see Recipes)
  21. 1x Hank’s Balanced Salt Solution (HBSS) (see Recipes)
  22. YPD agar plate (see Recipes)
  23.  Red blood cell (RBC) lysis buffer (see Recipes)
  24. 100% Percoll (see Recipes)
  25. 55%, 62%, and 81% Percoll (see Recipes)
  26. Heat-inactivated micrococcal nuclease (MNase, see Recipes)

Equipment

  1. FinnpipetteTM F1 Variable Volume Pipettes (Thermo Scientific, catalog numbers: 4641050N , 4641080N , and 4641100N )
  2.  Counting chamber Neubauer-improved (HAUSSER, catalog number: CB73811-01490 )
  3.  Autoclave Machine (see Note 2)
  4. 37 °C, 5% CO2 incubator (see Note 2)
  5. 30 °C incubator for growing C. albicans yeasts (see Note 2)
  6. Table top general-purpose centrifuge with swinging bucket rotor (KUBOTA, Model 4000, Rotor Name: ST-720M and PT-89M)
  7.  CellGard ES Energy Saver Class II Type A2 Biological Safety Cabinet (NuAire, model: NU-475-400 )
  8. Vortex-Genie 2 (Scientific Industries)
  9. Light Microscope (see Note 2)
  10. Surgical operating dissecting scissors (sharp/sharp) (see Note 2)
  11. Semken forceps (see Note 2)
  12. Euthanasia chamber that is used to administer CO2 for euthanasia (see Note 2)

Software

  1. Prism GraphPad Software (CA, USA)

Procedure

  1. Mouse bone marrow neutrophil enrichment 
    1. Mice at 6-12 weeks of age are placed in euthanasia chamber and euthanized by carbon dioxide for 4 min according to the AVMA Guidelines on Euthanasia (Cima, 2013). Obtain femur and tibia by using surgical operating dissecting scissors and Semken forceps.
      Note: All animal procedures and experimental protocols need to be approved by AAALAC-accredited facility of the host institute.
    2. Soak the bones in 1 ml ice cold 1x HBSS.
    3. Cut both ends of the bone, insert to one end the needle of a 3 ml syringe that is filled with ice cold 1x HBSS, push the plunger to flush out bone marrow cells into a 15 ml conical tube.
    4. Flush the bone one more time as in Step A3.
    5. Centrifuge the tube at 300 x g in room temperature for 10 min and discard the supernatant.
    6.  Resuspend cell pellet in 1 ml of ice cold RBC lysis buffer and leave it in room temperature for 1 min.
    7. Add 1 ml of 1x DPBS to the tube.
    8. Centrifuge the tube at 500 x g in room temperature for 5 min, discard the supernatant and suspend the pellet in 1 ml of 1x DPBS.
    9. Prepare three layers of discontinuous Percoll solutions (55%, 62%, and 81% Percoll in the order of top to bottom in a 15 ml conical tube. Each layer is consisted of 3 ml as shown in Figure 1).
    10. Overlay bone marrow cell suspension on top of the discontinuous Percoll gradient as shown in Figure 1.
    11. Centrifuge at 1,400 x g in room temperature for 30 min in a swing-out rotor, with the brake off.
    12. “Gently” remove the top 6 ml of the solution by micropipette and collect 1.0-1.5 ml of the solution containing the band of neutrophils at the interface of 62 and 81% as shown in Figure 1 to a new 15 ml conical tube.


    13. Figure 1. Enrichment of bone marrow neutrophils by discontinuous Percoll gradient centrifugation. Overlay bone marrow cell suspension on top of the 55/62/81% Percoll gradient (Step A10). After centrifugation in swing-out rotor (Step A11), there will be three visible bands in the gradient solution. Neutrophils are located at the interface of 62% and 81% Percoll (the lowest band). Carefully remove the top 6 ml of the solution and collect the top 1.0-1.5 ml of the remaining solution that contains cells at the lowest band (between the 2.5-4.0 mark on the scale) (Step A12).

    14. Add 8 ml of 1x DPBS to the tube containing neutrophils and centrifuge at 1,000 x g in room temperature for 5 min and discard the supernatant.
    15. Suspend the pellet in 2 ml of DPBS and centrifuge at 500 x g in room temperature for 3 min and discard the supernatant.
    16.  Suspend the pellet in 1 ml of HBSS.
    17. Dilute 10 µl of well-mixed cell suspension in 390 µl of 0.4% trypan blue solution and count the number of viable cells by loading 10 µl of the mixture to a Neubauer counting chamber. While viable cells are bright and shiny, dead cells stain blue. There is approximately 0.8 x 107-1 x 107 bone marrow cells per mouse.
    18.  Leave cells in room temperature until use.

  2. Preparation of fresh C. albicans 
    1. Thaw a frozen aliquot of C. albicans and plate it onto a YPD agar plate.
    2.  Incubate the dish at 30 °C overnight.
    3. Scrape C. albicans colonies with a sterile1,000 µl tip and streak the yeasts on YPD agar plate.
    4.  Incubate the plate at 30 °C overnight.
    5. Scrape C. albicans colonies with a sterile 200 µl tip and suspend in 1 ml HBSS.
    6. Take 10 µl of well-mixed C. albicans solution, dilute it in 390 µl of 4% trypan blue solution and count the number of viable yeast cells by loading 10 µl of the mixture to a Neubauer counting chamber. While viable yeast cells are bright and shiny, dead cells stain blue (approximately 0.5 x 107-2 x 107 C. albicans yeasts per ml).
    7. Leave C. albicans on ice until use.

  3. Neutrophils and C. albicans preparations
    1. Dilute bone marrow neutrophils obtained from Step A16 to 2 x 105 per 1 ml of HBSS solution.
    2.  Dilute freshly harvested C. albicans yeasts obtained from Step B7 to 4 x 105 per 1 ml of HBSS.
    3. Prepare 4 x 103 U/ml MNase by diluting 0.5 µl of enzyme stock in 250 µl of HBSS.

  4.  NET killing assay of C. albicans
    1. Seed 2 x 104 neutrophils (100 µl) in the wells of flat-bottomed 96-well plate and let adhere by incubation at 37 °C (5% CO2 incubator) for 30 min.
    2. Leave wells for 100 µl of HBSS without adding neutrophils as control as in Figure 2.
    3. Add 4 x 104 unopsonized C. albicans yeasts (100 µl) to all the wells (including control well) as shown in Figure 2.
    4. Add 0.5 µl of MNase (4 x 103 U/ml) at a final concentration of 10 U/ml or heat-inactivated MNase at otherwise equivalent concentration to appropriate wells as shown in Figure 2. Centrifuge the plate at 800 x g in room temperature for 3 min to spin down the yeasts.


    5. Figure 2. Layout for NET killing assay of C. albicans. Bone marrow neutrophils obtained from three different mice are suspended in 1x HBSS separately. Neutrophils from each mouse are seeded in two separate wells (Step D1). Leave one well for 1x HBSS only (Step D2). After incubation of the plate at 37 °C for 30 min, C. albicans suspensions are added to all three wells (Step D3). At the same time, MNase and heat-inactivated MNase (∆MNase) are added to separate wells separately (Step D4).

    6.  Incubate the plate at 37 °C (5% CO2 incubator) for 3 h.
    7. Add 0.5 µl of MNase to all the wells.
    8.  Leave the plate at 37 °C (5% CO2 incubator) for 15 min.
    9. Collect the content (about 200 µl) of each individual well into a microcentrifuge tube.
    10. Add 200 µl of ice cold H2O (pH = 11) to each well.
    11. Use mini cell scraper to scrape the bottom of each well followed by vigorous pipetting to dislodge C. albicans hyphae.
    12.  Collect the content of each well and add to the same microcentrifuge tubes.
    13. Repeat steps 9 to 11 once (total of 600 µl solution in the microcentrifuge tube now).
    14. Add 400 µl of HBSS to each microcentrifuge tube.
    15.  Vortex microcentrifuge tubes vigorously for 20 s.
    16. Make 1:10 serial dilutions of the supernatant and plate 100 µl of the diluted solution on YPD plate in duplicate.
    17. Seal the plates with paraffin and leave in 30 °C incubator for 2 days.
    18.  Enumerate colony forming units (CFUs) as shown in Figure 3.


      Figure 3. YPD plates with different C. albicans colony forming units (CFUs). Supernatants containing C. albicans are serially diluted (Step D15) and plated on YPD plates. The plates are incubated at 30 °C for 2 days (Step D16). C. albicans colonies are smooth and creamy white in color. There will be 3 different numbers of colonies from three 1:10 serially diluted supernatants. Select the plates with colonies fall between 30 and 300 (B) for counting and disregard the ones outside of this range (A). Carefully count each single colony even when they are very close to each other (B, red dotted circle).

Data analysis

  1. CFU of control well is N0; CFU of wells containing MNase is NM; CFU of wells containing heat-inactivated MNase is N∆M as shown in Figure 4.
    The average of CFU counts on the YPD dish in duplicate will be used as N0, NM and N∆M.
  2.  % of killing in the MNase group = (N0 - NM)/N0; % of killing in the heat-inactivated MNase group = (N0 - N∆M)/N0 as shown in Figure 4.


  3. Figure 4. Calculation of % NET killing of C. albicans. N0, NM and N∆M are determined by the average of CFU counts of duplicate control wells (C. albicans only), wells containing neutrophils, C. albicans, and MNase and wells containing neutrophils, C. albicans, and heat-inactivated MNase, respectively. % of killing is calculated as shown

  4. To estimate the % of yeast cells killed by NET, data are analyzed by Mann-Whitney test by comparing the % of killing in MNase group to that in heat-inactivated MNase group.

Notes

  1.  MNase digests NET. To include both MNase and inactivated MNase in Step D4 is to confirm that the killing is mediated by NET but not by other factors. To add MNase in Step D6 is to release NET-bound C. albicans from NET for subsequent plate count.
  2. The reagents and materials from the manufacturers listed are those that have been used for this assay in the authors’ laboratory. We have not tried reagents and materials from different manufacturers. The equipments that do not have their manufactures listed are common laboratory equipments.
  3. It is important to completely lyse red blood cells because RBC contamination may interfere NET formation. One should also note that repeating or prolonging RBC lysis in Step A6 may result in neutrophil death. Therefore, if 1 ml of ice cold RBC lysis buffer does not lyse all the RBCs, increase the volume but do not prolong the time for lysis.
  4. Germination-defective strain of C. albicans (e.g., HLC 54 strain) does not induce NET formation (Wu et al., 2019).
  5. Colony counts on YPD plates between 30 and 300 ensures accurate counting. Plating of several 1:10 serial dilutions of the supernatants collected in microcentrifuge tubes will ensure that plating one of the dilutions will result in counts within the range.

Recipes

  1. 1x Dulbecco's Phosphate Buffered Saline (DPBS)
    5 ml of 10x DPBS is diluted in 45 ml of ddH2O
  2. 1x Hanks' Balanced Salt Solution (HBSS)
    5 ml of 10x HBSS is diluted in 44.64 ml of ddH2O supplemented with 360 µl of sodium bicarbonate
  3. YPD agar plate
    1. Add 50 g of YPD broth and 20 g of agar A into 1 L of ddH2O
    2. Stir the solution until YPD is dissolved
    3.  Cool to 50 °C after autoclaving, and pour 23 ml of the medium into sterile 10-cm Petri dishes
    4. Store YPD dishes in refrigerator until use
  4. RBC lysis buffer
    1. 0.61 g of Tris-HCl and 4.15 g of NH4Cl are dissolved in 500 ml of ddH2O
    2.  Adjust the pH value to 7.4 and autoclave
  5. 100% Percoll
    Mix 45 ml of Percoll stock with 5 ml of 10x DPBS 
  6. 55%, 62%, and 81% Percoll
    55%, 62%, and 81% Percoll are prepared by diluting 100% Percoll (prepared in Recipe 5) with 1x DPBS
  7. Heat-inactivated MNase
    Heat MNase (4 x 103 U/ml) at 65 °C for 4 h and store in refrigerator until used

Acknowledgments

AcknowledgmentThis work was supported by Academia Sinica thematic project AS-105-TP-B08 to BWH and the Ministry of Science and Technology research grants 104-2320-B-002-052-MY2 and 107-2321-B-002-053-MY3 to BWH and SYW, respectively. This protocol was adapted from a publication by Vonk et al. (2012) and modified according to protocols that quantified the viability of biofilm-forming microorganisms in other studies ( Morici et al., 2016; Mohammed et al., 2017).

Competing interests

NO financial competing interests.

Ethics

Mouse study was carried out in strict accordance with the recommendations in the Guidebook for the Care and Use of Laboratory Animals, The Third Edition published by The Chinese-Taipei Society of Laboratory Animal Sciences in 2007. All animal procedures and experimental protocols were approved by AAALAC-accredited facility, the Committee on the Ethics of Animal Experiments of the National Taiwan University College of Medicine (Permit Number: 20140304, 20140533 and 20180013).

References

  1. Antachopoulos, C., Walsh, T. J. and Roilides, E. (2007). Fungal infections in primary immunodeficiencies. Eur J Pediatr 166(11): 1099-1117.
  2. 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.
  3. Chen, P. Y., Chuang, Y. C., Wang, J. T., Sheng, W. H., Yu, C. J., Chu, C. C., Hsueh, P. R., Chang, S. C. and Chen, Y. C. (2014). Comparison of epidemiology and treatment outcome of patients with candidemia at a teaching hospital in Northern Taiwan, in 2002 and 2010. J Microbiol Immunol Infect 47(2): 95-103.
  4. Cima, G. (2013). AVMA Guidelines for the Euthanasia of Animal: 2013 Edition. Javma-J Am Vet Med A 242: 715-716.
  5. Horn, D. L., Neofytos, D., Anaissie, E. J., Fishman, J. A., Steinbach, W. J., Olyaei, A. J., Marr, K. A., Pfaller, M. A., Chang, C. H. and Webster, K. M. (2009). Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry. Clin Infect Dis 48(12): 1695-1703.
  6. Kullberg, B. J. and Arendrup, M. C. (2015). Invasive Candidiasis. N Engl J Med 373(15): 1445-1456. 
  7.  Lionakis, M. S. and Netea, M. G. (2013). Candida and host determinants of susceptibility to invasive candidiasis. PLoS Pathog 9(1): e1003079.
  8. Mohammed, M. M. A., Pettersen, V. K., Nerland, A. H., Wiker, H. G. and Bakken, V. (2017). Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis. Anaerobe 44: 133-142.
  9. Morici, P., Fais, R., Rizzato, C., Tavanti, A. and Lupetti, A. (2016). Inhibition of Candida albicans biofilm formation by the synthetic lactoferricin derived Peptide hLF1-11. PLoS One 11(11): e0167470. 
  10. Vonk, A. G., Netea, M. G. and Kullberg, B. J. (2012). Phagocytosis and intracellular killing of Candida albicans by murine polymorphonuclear neutrophils. Methods Mol Biol 845: 277-287.
  11. Wu, S. Y., Huang, J. H., Chen, W. Y., Chan, Y. C., Lin, C. H., Chen, Y. C., Liu, F. T. and Wu-Hsieh, B. A. (2017). Cell intrinsic Galectin-3 attenuates neutrophil ROS-dependent killing of Candida by modulating CR3 downstream Syk activation. Front Immunol 8: 48.
  12.  Wu, S. Y., Weng, C. L., Jheng, M. J., Kan, H. W., Hsieh, S. T., Liu, F. T. and Wu-Hsieh, B. A. (2019). Candida albicans triggers NADPH oxidase-independent neutrophil extracellular traps through dectin-2. PLoS Pathog 15(11): e1008096.

简介

[摘要 ] 丰人病原体念珠菌白色念珠菌是顶级领先的原因之一全球总卫生保健相关血流感染。中性粒细胞是清除白色念珠菌感染的主要效应细胞。我们的研究表明,小鼠中性粒细胞利用两种独立的机制杀死白念珠菌:一种是CR3下游NADPH氧化酶依赖性机制,它可以杀死调理过的白色念珠菌。另一个是dectin-2介导的NADPH氧化酶非依赖性中性粒细胞胞外捕获物(NET),它杀死未调理的白色念珠菌。中性粒细胞杀死调理过的白色念珠菌 需要吞噬生物体并产生活性氧(ROS)。大多数现有的协议的测定中性粒细胞杀死白色念珠菌需要使嗜中性粒细胞吞噬生物体后洗涤步骤。根据定义,NET在细胞外杀死生物。因此,重要的是要跳过洗涤步骤,并向孔中添加最佳比例的嗜中性白细胞和白色念珠菌。为了证明NET的作用,有必要比较用微球菌核酸酶(MNase )(一种消化NET的酶)处理的嗜中性粒细胞的杀伤能力与用热灭活的MNase 处理的嗜中性粒细胞的杀伤能力。MNase 还用于释放与NET绑定的真菌元素以进行计数。该协议可用于测定其他生物膜形成生物的NET杀灭。

[背景 ] 念珠菌白色念珠菌是一种条件真菌病原体驻留作为粘膜表面上的共生,在大多数人类皮肤。温度,营养或血清的环境变化会导致其从酵母形式转化为菌丝。念珠菌感染是医疗中心和地区医院整体与卫生保健相关的血液感染的最主要诱因之一。即使接受抗真菌治疗,浸润性念珠菌病患者的死亡率也高达40%(Brown 等,2012;Chen 等,2014)。 ; Kullberg和Arendrup,2015年)。中性粒细胞减少和中性粒细胞功能障碍的患者极易发生侵袭性念珠菌病,这表明中性粒细胞抗念珠菌功能在宿主防御中很重要(Antachopoulos 等,2007;Horn 等,2009;Lionakis和Netea,2013)。我们的工作表明,小鼠嗜中性粒细胞利用两种独立的机制来杀死白色念珠菌,一种是NADPH氧化酶依赖性的,另一种是NADPH氧化酶依赖性的。调理的NADPH氧化酶-依赖性杀伤白色念珠菌需要由CR3介导的吞噬,和的NADPH氧化酶非依赖性杀伤unopsonized 白色念珠菌是通过树状-2介导的NET形成(吴等人,2017和2019)。MNase 是一种消化NET的酶。为了证明NET的作用,有必要将用MNase 处理的嗜中性粒细胞的杀伤能力与用热灭活的MNase 处理的嗜中性粒细胞的杀伤能力进行比较。两种处理的区别表示是被NET杀死,而不是被其他因素杀死(Wu 等,2019)。中性粒细胞是吞噬性的。通过受体吸收微生物会触发强大的ROS产生,从而杀死被吞噬的生物。为了测定NADPH氧化酶对调理白色念珠菌的杀灭作用,重要的步骤之一是在留出吞噬作用的时间后洗掉未吞噬的微生物(Vonk 等人,2012 ; Wu 等人,2017)。相比之下,由于NET会在细胞外杀死微生物,因此至关重要的是,不要洗掉未吞噬的生物体,以检测NET介导的白色念珠菌的杀灭。此外,不是用ddH 2 O在高pH(pH 11.0)下溶解嗜中性白细胞以释放被摄入的微生物,而是通过微型细胞刮刀和DNA消化酶MNase 从孔中分离出形成NET的嗜中性白细胞和真菌菌丝元素(Wu 等。,2017年和2019年)。此处描述的方法于2019年发表(Wu 等,2019),以观察NET 对白色念珠菌的杀灭作用。该方法可适用于量化NET介导的其他生物膜形成生物的杀灭。

关键字:中性粒细胞胞外诱捕, 白色念珠菌, 中性粒细胞, 抑菌活性, 微球菌核酸酶

材料和试剂


 


1. P ipette提示      


2. 赠送细胞刮刀(毕天火,目录号:22003)      


3. 平底96孔板(Corning,目录号:3599 )      


4. 15 ml锥形管(Corning,目录号:430791)      


5. 90毫米x 15毫米培养皿(Alpha Plus,目录号:16001 )      


6. 1.5米升微量离心管(Corning公司,目录号:MCT-150-C)      


7. 3毫升带23号针头的注射器(BD,目录号:302111 )      


8. 封口膜(Bemis,目录号:PM996)      


9. 白色念珠菌SC5314菌株(ATCC MYA-2876)      


10. 6-12周龄的C57BL / 6JNarl bacground中的雌性近交小鼠(国家实验动物中心,RMRC11005)   


11. Percoll (GE H ealthcare,目录号:17-0891-01)   


12. 10倍Dulbecco磷酸盐缓冲盐水(D PBS)(生物工业,目录号:02-023-1A)   


13. 10倍汉克平衡盐溶液(HBSS)(生物工业,目录号:02-015-5A)   


14. 0.4%台盼蓝(Sigma-Aldrich,目录号:93595)   


15. 7.5 %碳酸氢钠(Biological Industries,目录号:03-040-1B)   


16. 酵母-p- 右旋糖(YPD)肉汤(Bioshop ,目录号:YPD002.500)   


17. 琼脂A(BIO BASIC,目录号:FB0010)   


18. 2 x 10 6 U / ml微球菌核酸酶(MNase )(NEB ,目录号:M0247S )   


19. 无菌双蒸馏水H 2 O(ddH 2 O),通过NaOH调节至pH 11   


20. 1x Dulbecco磷酸盐缓冲盐水(DPBS)(请参阅食谱)   


21. 1x Hank的平衡盐溶液(HBSS)(请参阅食谱)   


22. YPD琼脂平板(请参阅食谱)   


23. 红细胞(RBC)裂解缓冲液(请参阅食谱)   


24. 100%Percoll (请参阅食谱)   


25. 55%,62%和81%Percoll (请参阅食谱)   


26. 热灭活的微球菌核酸酶(MNase ,参见食谱)   


 


设备


 


Finnpipette TM F1可变体积导管(Thermo Scientific,目录号:4641050N,4641080N和4641100N)
计数室经过Neubauer改进(HAUSSER,目录号:CB73811-01490)
高压釜机(见注2)
37°C,5%CO 2 培养箱(见注2)
30°C培养箱,用于培养白色念珠菌酵母(请参见注释2)
台式通用离心机,带有旋转斗式转子(KUBOTA,4000型,转子名称:ST-720M和PT-89M)
CellGard ES节能II类A2型生物安全柜(NuAire ,型号:N U-475-400)
Vortex-Genie 2(科学产业)
光学显微镜(见注2)
手术解剖剪刀(锋利/锋利)(请参阅注释2)
塞姆肯钳(见注2)
Eu的是,用于管理CO thanasia室2 安乐死(见注2)
 


软件


 


Prism GraphPad软件(美国加利福尼亚)
 


程序


 


小鼠骨髓中性粒细胞富集
将6-12周龄的小鼠置于安乐死室中,并根据《 AVMA安乐死指南》(Cima,2013)用二氧化碳安乐死4分钟。使用手术解剖剪刀和塞姆肯钳获取股骨和胫骨。
注意:所有动物操作规程和实验规程都必须经过AAALAC认可的寄主机构的批准。


将骨头浸入1毫升冰冷的1x HBSS中。
切开骨头的两端,一端插入装有冰冷的1x HBSS 的3 ml 注射器的针头,推动柱塞将骨髓细胞冲洗入15 ml锥形管中。
冲洗骨一个更多的时间如在小号TEP 甲3。
在室温下以300 x g 离心管10分钟,并弃去上清液。
将细胞沉淀重悬于1 ml冰冷RBC裂解缓冲液中,并在室温下放置1分钟。
向管中加入1 ml 1x D PBS。
将试管在室温下以500 x g的速度离心5分钟,弃去上清液并将沉淀物悬浮在1 ml的1x D PBS中。
准备三层不连续的Percoll 溶液(在15 ml的锥形管中,按从上到下的顺序依次排列55%,62%和81%Percoll 。每层由3 ml组成,如图1所示)。
在不连续的Percoll 梯度上覆盖骨髓细胞悬浮液,如图1所示。
在室温下以1400 x g的转速在可旋转的转子中离心30分钟,并关闭制动器。
如图1所示,用微量移液器“轻轻地”除去顶部的6毫升溶液,并在新的15毫升锥形管中收集1.0-1.5毫升在62%和81%界面处含有嗜中性白带的溶液。
 


D:\重新格式化\ 2020-6-1 \ 2003080--1475 Betty Wu-Hsieh 850860 \图jpg \图1.jpg


图1。通过不连续的Percoll 梯度离心富集骨髓中性粒细胞。将骨髓细胞悬液覆盖在55/62/81%Percoll 梯度的顶部(步骤A 10)。在摆出的转子中离心(步骤A 11)后,梯度溶液中将出现三个可见带。中性粒细胞位于62%和81%Percoll (最低谱带)的界面。小心地取出前6毫升该溶液并收集顶部1.0-1.5毫升含有细胞在最低频带中的剩余溶液(2.5之间- 在标尺4.0马克)(步骤A 12)。


 


向装有中性粒细胞的试管中加入8 ml 1x D PBS,在室温下以1,000 x g 离心5分钟,弃去上清液。
小号uspend在2ml的沉淀d PBS和离心机在500 X 克在室温下3分钟并弃去上清液。
将沉淀物悬浮在1 ml HBSS中。
在390微升的稀释10 OFL良好混合的细胞悬浮液的0.4 %台盼蓝溶液和计数的数量通过加载10μl的该混合物的活细胞的纽鲍尔计数室。活细胞明亮有光泽,而死细胞则染成蓝色。有approxi 三方共同0.8×10 7 - 1 X 10 7 每只小鼠骨髓细胞。
将细胞置于室温下直至使用。
 


的新鲜制剂C. 白色念珠菌
解冻冷冻的白色念珠菌的等分试样,并将其铺在YPD琼脂平板上。
将培养皿在30°C下孵育过夜。
刮白色念珠菌菌落用sterile1 ,000 TIPL 尖端和条纹在YPD酵母琼脂平板上。
在30°C下孵育平板过夜。
刮白色念珠菌用无菌200个菌落TIPL类型并悬浮于1ml HBSS。
取10毫升充分混合的白色念珠菌溶液,将其在390微升4%台盼蓝溶液中稀释,并通过将10微升混合物装入Neubauer 计数室来控制活酵母细胞的数量。而存活酵母细胞是明亮而有光泽,死细胞染成蓝色(AP 邻近地0.5×10 7 - 2 X 10克7 的白色念珠菌酵母每毫升)。
将白色念珠菌放在冰上直至使用。
 


中性粒细胞和白色念珠菌制剂
每1 ml HBSS溶液将从S tep A 16 获得的骨髓中性粒细胞稀释至2 x 10 5 。
将从S tep B 7 获得的新鲜收获的白色念珠菌酵母稀释至每1 ml HBSS 4 x 10 5 。
通过在250 µl HBSS中稀释0.5 ofl 的酶储备液来制备4 x 10 3 U / ml MNase 。
 


白色念珠菌的净杀灭测定
在平底的96孔板的孔中接种2 x 10 4个中性粒细胞(100 µl),并通过在37°C(5%CO 2 培养箱)中孵育30分钟使其粘附。
如图2所示,在不添加嗜中性粒细胞的情况下,留出100份HBSS的孔。
如图2所示,向所有孔(包括对照孔)中添加4 x 10 4个非调理的白色念珠菌酵母(100 µl)。
加入0.5微升的MNase (4 X 10 3 单位/毫升)以10单位/毫升或热失活的最终浓度MNase 在否则当量浓度至适当的孔中,如图2Ç entrifuge将板在800 X 克在在室温下放置3分钟,以使酵母发酵。
 


D:\重新格式化\ 2020-6-1 \ 2003080--1475 Betty Wu-Hsieh 850860 \图jpg \图2.jpg


图2. 白色念珠菌NET杀死试验的布局。从三只不同的小鼠获得的骨髓中性粒细胞分别悬浮在1x HBSS中。将来自每只小鼠的嗜中性粒细胞浸入两个单独的孔中(步骤D 1)。为1x HBSS留一口井(步骤D 2)。之后将板在37温育° 下进行30分钟,白色念珠菌悬浮液一个dded所有三个孔中(步骤d 3)。同时,将MNase 和热灭活的MNase (∆ MNase )分别添加到胎次孔中(步骤D 4)。


 


在37°C(5%CO 2 培养箱)中将板孵育3小时。
向所有孔中加入0.5 毫升的MNase 。
将板在37°C(5%CO 2 培养箱)中放置15分钟。
将每个孔的内容物(约2 00 µl )收集到微量离心管中。
向每个孔中加入200毫升冰冷的H 2 O(pH = 11)。
使用微型细胞刮刀刮擦每个孔的底部,然后用力吹打除去白色念珠菌菌丝。
收集每个孔的内容物,并添加到相同的微量离心管中。
重复步骤9至11(现在在微量离心管中总共有600个溶液)。
向每个微量离心管中加入400毫升HBSS。
剧烈涡旋微量离心管20 s。
对上清液进行1:10连续稀释,并在YPD平板上一式两份地稀释100份稀释溶液。
用石蜡密封板,并在30°C 和培养箱中放置2天。
枚举菌落形成单位(CFU),如图3所示。
 


D:\重新格式化\ 2020-6-1 \ 2003080--1475 Betty Wu-Hsieh 850860 \图jpg \图3.jpg


图3.具有不同白色念珠菌菌落形成单位(CFU)的YPD板。连续稀释含有白色念珠菌的上清液(步骤D15),并铺在YPD板上。将板在30℃温育 ℃ 2天(步骤D16)。白色念珠菌菌落颜色光滑且呈乳白色。从三个1:10连续稀释的上清液中将有3个不同数量的菌落。选择菌落在30到300之间的板(B)进行计数,而忽略该范围以外的板(A)。即使每个菌落彼此非常靠近,也要仔细计数(B,红色虚线圆圈)。


 


数据分析


 


控制井的CFU为N 0 ; 含有MNase 的孔的CFU 为N M ; 含有热灭活的MNase 的孔的CFU 为N ∆ M ,如图4所示。
平均在YPD碟和重复的CFU计数的将被用于如N 0 ,N,中号和N Δ 中号。


MNase 组的杀死百分率=(N 0 -N M )/ N 0 ; 如图4所示,热灭活的MNase 组中的杀死百分比=(N 0 -N ∆ M )/ N 0 。
 


D:\重新格式化\ 2020-6-1 \ 2003080--1475 Betty Wu-Hsieh 850860 \图jpg \图4.jpg


图4. 白色念珠菌净杀灭%的计算。N 0 ,N M 和N ∆ M 由重复对照孔(仅白色念珠菌),含有中性粒细胞,白色念珠菌和MNase的孔以及包含中性粒细胞,白色念珠菌和加热的孔的CFU计数的平均值确定。灭活的MNase 。如下所示计算杀戮百分比


 


为了估计由NET杀死酵母细胞的%,数据由Mann-Whitney检验,通过比较在杀死%分析MNase 在热失活的组到该MNase 基。
 


笔记


 


MNase 消化NET。在步骤D 4中包括MNase 和灭活的MNase 都是为了确认杀伤是由NET介导的,而不是由其他因素介导的。在步骤D 6中添加MNase的目的是从NET中释放与NET绑定的白色念珠菌,以用于随后的平板计数。
所列制造商的试剂和材料是作者实验室中用于该测定的试剂和材料。我们尚未尝试使用不同制造商的试剂和材料。该设备没有自己的制成品列出的是常见的实验室设备。
完全溶解红细胞很重要,因为RBC污染可能会干扰NET的形成。还应注意,在步骤A 6 中重复或延长RBC裂解可能会导致嗜中性粒细胞死亡。因此,如果1 ml冰冷的RBC裂解缓冲液不能裂解所有RBC,则增加体积但不延长裂解时间。
的发芽缺陷株白色念珠菌(例如,HLC 54株)不诱导NET形成(吴等人,2019)。
YPD平板上的菌落计数在30到300之间,可确保准确计数。对微量离心管中收集的上清液的几种1:10连续稀释液进行电镀将确保电镀其中一种稀释液将导致计数在此范围内。
 


菜谱


 


1x Dulbecco磷酸盐缓冲盐水(D PBS)
将5 ml的10x DPBS稀释在45 ml的ddH 2 O中


1x Hanks的平衡盐溶液(HBSS)
5毫升10X HBSS中44.64毫升的双蒸水稀释2 ö补充有360 微升的钠BICAR bonate


YPD琼脂板
将50克YPD肉汤和20克琼脂A加入1升ddH 2 O中
搅拌溶液直至YPD溶解
高压灭菌后冷却至50°C,然后将23 ml培养基倒入无菌的10 cm培养皿中
将YPD餐具存放在冰箱中直至使用
RBC裂解缓冲液
将0.61克Tris-HCl和4.15克NH 4 Cl溶解在500毫升ddH 2 O中
调节pH值至7.4并高压灭菌
100%Percoll
将45 ml Perco ll 储备液与5 ml 10x DPBS混合


55%,62%和81%Percoll
通过用1x D PBS 稀释100 %Percoll (在配方5中制备)来制备55%,62%和81%的Percoll。


热灭活的MNase
在65 °C下将MNase (4 x 10 3 U / ml )加热4小时并储存在冰箱中直到使用。


 


致谢


 


这项工作得到了BWH的中央研究院主题项目AS-105-TP-B08的支持,科学技术部的研究补助金为104-2320-B-002-052-MY2和107-2321-B-002-053-MY3分别为BWH和SYW。该协议改编自Vonk 等人的出版物。(2012年),并根据在其他研究中量化生物膜形成微生物生存力的方案进行了修改(Morici 等,2016;Mohammed 等,2017)。


 


利益争夺


 


没有财务竞争利益。


 


伦理


 


严格按照《实验动物的护理和使用指南》(第三版)的要求进行了小鼠研究,该实验手册由中华台北实验动物科学学会于2007年出版。所有动物程序和实验方案均获得AAALAC的批准国立台湾大学医学院动物实验伦理委员会认可的机构(许可证编号:20140304、20140533和20180013)。


 


参考文献


 


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Chen PY,Chuang,YC,Wang,JT,Sheng,WH,Yu,CJ,Chu,CC,Hsueh,PR,Chang,SC and Chen,YC(2014)。2002年和2010年在台湾北部的一家教学医院中对念珠菌血症患者的流行病学和治疗结果的比较。《微生物免疫感染》(J),《微生物感染》47(2):95-103。
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Mohammed,MMA,Pettersen,VK,Nerland ,AH,Wiker ,HG和Bakken,V.(2017年)。从核镰刀菌和牙龈卟啉单胞菌的单物种和双物种生物膜中提取的细胞外基质的定量蛋白质组学分析。厌氧菌44:133-142。
Morici ,P.,Fais ,R.,Rizzato ,C.,Tavanti ,A.和Lupetti ,A.(2016)。合成乳铁蛋白衍生肽hLF1-11 对白色念珠菌生物膜形成的抑制作用。PLoS One 11(11):e0167470。              
Vonk AG,Netea ,MG和Kullberg,BJ(2012)。鼠多形核中性粒细胞吞噬白色念珠菌并吞噬细胞。方法分子生物学845:277-287。
伍锡永,黄建华,陈文怀,陈玉元,林春水,陈玉江,刘FT,吴学熙(2017)。细胞内在的Galectin-3可通过调节CR3下游Syk激活来减弱念珠菌对中性粒细胞ROS的杀伤作用。前台免疫8:48 。
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引用:Wu, S. and Wu-Hsieh, B. A. (2020). Neutrophil Extracellular Trap Killing Assay of Candida albicans . Bio-protocol 10(16): e3716. DOI: 10.21769/BioProtoc.3716.
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