参见作者原研究论文

本实验方案简略版
Apr 2020
Advertisement

本文章节


 

Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay
用CCF4-AM FRET法检测RAW264.7巨噬细胞中海洋分枝杆菌的细胞质移位   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, the substrate of β-lactamase. The exogenously added, neutral charged-FRET reagent can diffuse across the membrane and stay in the cytosol only once it is charged in the cytosol. When bacteria translocate from subcellular organelles (e.g., phagosomes) to the cytosol, the bacteria-associated β-lactamase cleaves the β-lactam ring, resulting in loss of FRET signal. Here we describe the fluorometer-based approach optimized for direct measurement of cytosolic translocation as a result of the EsxAB complex of Mycobacterium marinum in RAW264.7 cells.

Keywords: CCF4-AM (CCF4-AM), Mycobacterium marinum (海洋分支杆菌), EsxA (EsxA), EsxB (EsxB), FRET (荧光共振能量转移)

Background

The development of a β-lactamase reporter assay gave rise to the subsequent Förster resonance energy transfer (FRET) assay (Zlokarnik et al., 1998; Cavrois et al., 2002). Two fluorescence dyes, hydroxycoumarin (donor) and fluorescein (acceptor), are linked via a CCF4-AM (CCF2-AM in earlier reports) β-lactam ring. When excited at 405 nm, hydroxycoumarin emits at 450 nm (blue), which subsequently excites fluorescein to emit at 527 nm (green). Cleavage of the β-lactam ring by bacteria-associated β-lactamase results in loss of FRET and direct emission of hydroxycoumarin (blue) (Jones and Padilla-Parra, 2016). The CCF4-AM FRET reporter assay is versatile and has been been used for a variety of different applications, such as tracking molecular fusions (Jones and Padilla-Parra, 2016; Shaikh, et al., 2019), tracking delivery of biological cargos to the cytosol (Stone et al., 2018), and monitoring translocation of bacteria within cells (Charpentier and Oswald, 2004). Since Mycobacterium marinum also possesses β-lactamase (Flores et al., 2005), Simeone and colleagues exploited the FRET assay to track cytosolic translocation of Mycobacterium tuberculosis in THP-1 cells (Simeone et al., 2012). Recently, we applied this method for investigation of the role of EsxAB in cytosolic translocation of Mycobacterium marinum (Acosta et al., 2014; Zhang et al., 2016; Aguilera et al., 2020). RAW264.7 cells were infected with M. marinum and the results were obtained under a fluorometer, providing a quick, quantitative method for analysis of results. Furthermore, as Mycobacterium marinum is a BSL-2 pathogen and contains similarities in its ESX-1 locus (Tobin and Ramakrishnan, 2008), it provides a safe, direct way to investigate the EsxAB complex. Here we describe our procedure for direct monitoring of cytosolic translocation of Mycobacterium marinum in RAW264.7 cells via the CCF4-AM FRET assay using a fluorometer.

Materials and Reagents

  1. Inoculation loop (BD, catalog number: 220216)
  2. Cell Scraper (Fisherbrand, catalog number: 08-100-241)
  3. T-75 Flask (Thermo Scientific, catalog number: 12-566-440)
  4. 50 ml Centrifuge tube (Corning, catalog number: 352098)
  5. 6-well plate (Corning, catalog number: 3516)
  6. 10 ml syringe (Fisherbrand, catalog number: 14-955-459)
  7. 22 gauge needle (BD, catalog number: 305900)
  8. 27 gauge needle (BD, catalog number: 305109)
  9. 5 µm filter (Millex, catalog number: SLSV025LS)
  10. Pasteur pipette (Fisherbrand, catalog number: 13-678-6b)
  11. Aluminum Foil (Fisherbrand, catalog number: 01-213-102)
  12. Thin Wall Cuvette (Fireflysci, catalog number: 30FL)
  13. 435 nm long-pass filter (Andover Corporation, catalog number: 435FG03-50S)
  14. 7H9 media (Difco, catalog number: 271310)
  15. OADC Enrichment (BD, catalog number: 211886)
  16. Tween-80 (Sigma-Aldrich, catalog number: P1754)
  17. Kanamycin Monosulfate (Gold Bio, catalog number: K-120-5)
  18. DMEM media (Gibco, catalog number: 10566-016)
  19. Fetal Bovine Serum (Gibco, catalog number: A4766801)
  20. Penicillin and Streptomycin (Gibco, catalog number: 15070063)
  21. Phosphate buffered saline (Sigma-Aldrich, catalog number: P3813)
  22. CCF4-AM FRET Loading Solutions (Life Technologies, catalog number: K1085)
    Note: This kit contains Solution A, Solution B, and Solution C as described in the procedure.
  23. DMSO (Sigma-Aldrich, catalog number: D8418)
  24. 4% Paraformaldehyde
  25. NaCl (Fisherbrand, catalog number: 18-606-420)
  26. MgCl (Fisherbrand, catalog number: 18-604-047)
  27. CaCl2 (Fisherbrand, catalog number: AA4428030)
  28. KCl (Spectrum Chemical, catalog number: 18-605-507)
  29. Glucose (Sigma-Aldrich, catalog number: G7021)
  30. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid (HEPES)) (Fisher Bioreagents, catalog number: BP310)
  31. Tris (Fisher Bioreagents, catalog number: BP152-500)
  32. EM Media (see Recipes)

Equipment

  1. Biosafety Cabinet (Labconco) 
  2. Vortex (VWR, catalog number: 58816-121)
  3. Hemocytometer (Hausser Scientific, catalog number: S17040)
  4. Inverted Light Microscope (VWR, catalog number: Vistavision)
  5. Cell Imaging System (Life Technologies, catalog number: 4471136)
  6. Spectrophotometer (Amersham Biosciences, model: Ultrospec 10)
  7. Table Top Centrifuge (Eppendorf, catalog number: 5810R)
  8. CO2 Incubator (VWR, catalog number: 3078)
  9. Fluorometer (ISS, catalog number: K2)
  10. Incubator Shaker (VWR, catalog number: 1585)
  11. Hot Plate/Stirrer (ThermoFisher Scientific, catalog number: SP131325)
  12. pH meter (Mettler Toledo, catalog number: 30266626)

Procedure

  1. Growth of M. Marinum bacterial culture
    1. Approximately 5 d before the infection assay, prepare the M. Marinum culture for infection. 
    2. Inoculate M. marinum into 50 ml of 7H9 media supplemented with 10% OADC enrichment and proper antibiotics as needed, we routinely use 30 mg/ml Kanamycin. 
      1. This bacterial culture will be used to start a new bacterial culture and passaged to reduce cell clumping. 
      2. If the bacterial culture contains too many clumped cells, then add Tween-80 at a concentration of 0.05% (v/v) to the 7H9 media. 
    3. Allow the bacterial culture to grow at 30 °C while shaking at 150 rpm until it reaches approximately OD600 1.2.
    4. Once the bacterial culture has grown past mid log phase, transfer 4 ml of culture into 46 ml of fresh 7H9 media supplemented with 10% OADC.
    5. Incubate at 30 °C while shaking at 150 rpm; allow the bacterial culture to grow into mid log phase once more, approximately 2 d.

  2. Culturing of RAW264.7 mouse macrophage cells
    1. Approximately 2-3 d before infection, grow RAW264.7 cells in a T-75 flask with 10 ml of DMEM media containing 10% Fetal Bovine Serum (FBS) with penicillin and streptomycin at 100 unit/ml. 
    2. Allow the cells to grow to 80-90% confluence at 37 °C with 5% CO2
    3. The day before the infection, use a cell scraper to lift all of the cells (at 80-90% conflucence) from the  bottom of the TC flask.
    4. Transfer the cells and media into a 50 ml centrifuge tube.
    5. Centrifuge at 160 × g for 10 min at RT. 
    6. Remove supernatant media and discard.
    7. Resuspend the pellet in 10 ml of DMEM media containing 10% FBS with penicillin and streptomycin. 
    8. Count the cells using whichever method you prefer. Record the approximate number of cells in the sample. 
    9. Fill a 6-well plate with 2 ml of media per well, then add RAW264.7 cells to seed a total of 2.5 × 106 cells/well.
    10. Incubate the cells overnight at 37 °C with 5% CO2.

  3. Bacterial single-cell preparation 
    1. Pellet down the bacterial culture from Part A by centrifuging at 3,220 × g for 15 min. 
    2. Remove the supernatant and resuspend the bacteria in 50 ml of PBS. 
    3. Wash the cells 2 more times for a total of 3 washes with PBS. 
    4. After the third wash, remove the supernatant and resuspend the pellet in 10 ml of PBS. 
    5. Aspirate the bacteria in PBS using a syringe and pass through a 22-gauge needle.
      Note: This step is optional but will facilitate passing the culture through a filter. 
    6. Aspirate the bacteria with a syringe and pass through a 27-gauge needle. 
    7. Aspirate the bacteria and pass through a 5 µm filter. 
    8. Measure the density of the bacterial culture with OD600
    9. Calculate the volume of bacterial cells needed to add to your macrophages depending on the multiplicity of infection (MOI). 
    10. If needed, concentrate the bacterial cells by pelleting and resuspending them into a smaller volume.

  4. Infection of macrophages
    1. Check RAW264.7 cells under a microscope; they should be at 60% confluence. 
    2. Aspirate DMEM media and replace with EM medium. 
    3. Infect macrophages with Mycobacterium marinum at 10 MOI, or at the desired MOI. 
    4. Incubate for 2 h at 30 °C with 5% CO2.
    5. After incubation, aspirate the media. 
    6. Gently wash with 3 ml PBS and agitate the plate.
    7. Remove the PBS and wash 2 more times to remove extracellular bacteria.
    8. Add 2.5 ml of pre-warmed EM media with 10% FBS and incubate for 48 h.

  5. CCF4-AM FRET Assay
    1. Load RAW264.7 cells with CCF4-AM loading solutions by following the manufacturer’s recommendation:
      1. The uninfected RAW264.7 cells loaded with CCF4-AM serve as a negative control. 
      2. The RAW264.7 cells without CCF4-AM serve as another negative control. 
      3. A Chiense hamster ovary (CHO) cell line that expresses β-lactamase (CHO-β-Lac) is used as a positive control for cleavage of CCF4-AM (Zhang et al., 2016).
      4. Solution B (100 mg/ml Pluronic-F127 surfactant in DMSO and 0.1% acetic acid) and Solution C (24% w/w PEG 400, 18% TR-40 v/v in water) are prepared by manufacturer. 
    2. Add 182 µl of DMSO to dissolve 200 µg of CCF4-AM, gently pipet up and down.
      Note: This will be solution A, as specified by manufacturer. 
    3. Decant the solution into 20 µl aliquots.
      Notes:
      1. If you wish to store, desiccate under vacuum and store at -20 °C protected from light. 
      2. If vacuum is not available, the solution may be stored at -20 °C in the dark. This solution is stable for up to three months. 
    4. Add 3 µl of Solution A to 30 µl of Solution B and vortex. 
    5. Add 467 µl of Solution C and vortex to produce 6× CCF4-AM substrate loading solution.
      Note: This solution is stable for 12 h at RT. 
    6. Add 500 µl of 6× CCF4-AM loading solution to 100 µl of the cell culture to make 1× CCF4-AM loading solution. 
    7. Incubate at room temperature for 2 h while protected from light.
      Note: If cells are disturbed, the 2-h incubation will give them enough time to settle to the bottom of the plate. 
    8. Aspirate the media and CCF4-AM substrate.
    9. Resuspend the cells with 3 ml PBS and agitate the plate gently.
    10. Remove the PBS and wash 2 more times with PBS for a total of 3 washes.
    11. Fix the cells with fresh 4% paraformaldehyde (PFA) for 30 min at room temperature (RT) while protected from light.
    12. Wash once more with PBS and at this stage the cells are ready for measurement.

  6. Measurement using a fluorometer
    1. Gently resuspend the cells in PBS and transfer them to a thin walled UV cuvette. 
    2. The fluorometer program should be set up to excite at 405 nm and record emissions between 430 nm to 600 nm to obtain the curves containing both blue and green peaks (Figure 1).
      Note: In the fluorometer, two polarizers are placed vertical to each other on the excitation beam path and a 435 nm long-pass filter is placed on the emssition beam path to reduce the background and scattered light.



      Figure 1. The emission spectra of the samples were recorded from FRET measurement. A. After excitation at 405 nm, the hydroxycoumarin will transfer energy to fluorescein resulting in a green emission at 527 nm. Bacteria containing β-lactamase will escape the phagosome and come into contact with the FRET dye in the cytosol, resulting in the loss of FRET and a blue emission at 450 nm. B. The first peak is the emission of hydroxycoumarin (blue) and the second peak is the emission of fluorescein (green).

    3. The intensitiy at 450 nm (blue), I1, and the intensity at 527 nm, I2, are measured for 20 iterations. The Blue/Green ratio is calculated as I1/I2 (Figure 2).


      Figure 2. The blue/green ratio for sample data is calculated by division of I1 by I2. The intensity at 450 nm (blue) and 527 nm (green) are measured for 20 iterations. The Blue/Green ratio is calculated. A higher ratio, as observed for Mycobacterium marinum of ~0.8 represents a good change of signal, indicating cytosolic translocation. Lower ratios (< 0.2) are obtained by bacteria unable to escape the phagosome or the negative control.

Data analysis

  1. The Blue/Green ratios are averaged for 20 iterations from 3 duplicates, a total of 60 replicates. 
  2. The average Blue/Green ratio is then plotted as a bar graph with error bars representing standard deviation and tested for statistical significance (Figure 3).


    Figure 3. Sample data as bar graph with Blue/Green ratio for different strains. By plotting the data in a bar graph, the loss of FRET between samples can be seen. In this scenario, the wild type M. marinum escapes the phagosome and interacts with the FRET dye resulting in a higher Blue/Green ratio. The M. marinumΔEsxA/B is unable to escape the phagosome and does not interact with the FRET dye resulting in no change of color from green to blue.

  3. The values are tested for normality using the Shapiro-Wilk test.
  4. If the data fails to differ from normal, it is tested for significance with a One-way ANOVA, otherwise a Tukey-Kramer test is performed to test for statistical significance.

Recipes

  1. EM Media (1 L)
    1. In a beaker add 7.02 g of NaCl, 0.52 g of KCl, 0.26 g of CaCl2, 0.076 g of MgCl, 0.9 g glucose and 6.5 of HEPES to water
    2. Bring the pH to 7.3
    3. Bring water up to 1 L

Acknowledgments

The study is supported by the grants from NIGMS (SC1GM095475 to J. Sun), National Center for Research Resources (5G12RR008124) and National Institute on Minority Health and Health Disparities (G12MD007592). Javier Aguilera was supported by National Institutes of Health Grant (R25GM069621 to R. Aguilera), via the RISE program for graduate students. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Competing interest

The authors have no competing interests to report.

References

  1. Acosta, Y., Zhang, Q., Rahaman, A., Ouellet, H., Xiao, C., Sun, J., and Li, C. (2014). Imaging cytosolic translocation of Mycobacteria with two-photon fluorescence resonance energy transfer microscopy. Biomed Opt Express 5(11): 3990-4001.
  2. Aguilera, J., Karki, C. B., Li, L., Reyes, S. V., Estevao, I., Grajeda, B. I., Qi Zhang, Arico, C. D., Ouellet, H. and Sun, J. (2020). Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence. J Biol Chem 295(17): 5785-5794.
  3. Cavrois, M., de Noronha, C., and Greene, W. C. (2002). A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20(11): 1151-1154.
  4. Charpentier, X. and Oswald, E. (2004). Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 β-lactamase as a new fluorescence-based reporter. J Bacteriol 186(16): 5486-5495.
  5. Flores, A. R., Parsons, L. M., and Pavelka Jr, M. S. (2005). Genetic analysis of the β-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to β-lactam antibiotics. Microbiology 151(2): 521-532.
  6. Jones, D. M., and Padilla-Parra, S. (2016). The β-lactamase assay: Harnessing a FRET biosensor to analyse viral fusion mechanisms. Sensors 16(7): 950.
  7. Simeone, R., Bobard, A., Lippmann, J., Bitter, W., Majlessi, L., Brosch, R., and Enninga, J. (2012). Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS Pathog 8(2): e1002507.
  8. Shaikh, F., Zhao, Y., Alvarez, L., Iliopoulou, M., Lohans, C., Schofield, C. J., Parra, S. P., Siu, S. W. I., Fry, E. E. and Ren, J. S. (2019). Structure-Based in silico screening identifies a potent ebolavirus inhibitor from a traditional chinese medicine library. J Med Chem 62(6): 2928-2937.
  9. Stone, S. R., Heinrich, T., Juraja, S. M., Satiaputra, J. N., Hall, C. M., Anastasas, M., Mills ,D. M., Chamberlain, C. A., Winslow, S. and Priebatsch, K. (2018). β-Lactamase tools for establishing cell internalization and cytosolic delivery of cell penetrating peptides. Biomolecules 8(3): 51.
  10. Tobin, D. M., and Ramakrishnan, L. (2008). Comparative pathogenesis of Mycobacteriummarinum and Mycobacterium tuberculosis. Cell Microbiol 10(5): 1027-1039.
  11. Zhang, Q., Wang, D., Jiang, G., Liu, W., Deng, Q., Li, X., Qian, W., Ouellet, H. and Sun, J. (2016). EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5. Sci Rep 6: 32618.
  12. Zlokarnik, G., Negulescu, P. A., Knapp, T. E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K. and Tsien, R. Y. (1998). Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter. Science 279(5347): 84-88.

简介


[摘要]的CCF4-AM福斯特共振能量转移(FRET)测定法是测量活细胞中的细菌胞质转运的敏感的方法。FRET对羟基香豆素(供体)和荧光素(受体)是通过连接一个CCF4-AMβ内酰胺环,β内酰胺酶的底物。外源添加的中性带电FRET试剂可以扩散到整个膜中,并且只有在被充入细胞溶质后才停留在细胞溶质中。当细菌从亚细胞器(例如吞噬体)转运到细胞质时,细菌相关的β-内酰胺酶会裂解β-内酰胺环 ,导致FRET信号丢失。在这里,我们描述了荧光-基础的方法胞质易位的直接测量作为优化的结果EsxAB复杂的分枝海鱼在RAW264.7细胞。



[背景技术]开发一个β内酰胺酶报道基因测定的产生了后续的福斯特共振能量转移(FRET)测定法(Zlokarnik等人,19。9 8; Cavrois等人。,2002)。两种荧光染料羟基香豆素(供体)和荧光素(受体)通过CCF4-AM(在较早的报道中为CCF2-AM)β-内酰胺环连接。当在405 nm处激发时,羟基香豆素在450 nm处发射(蓝色),随后激发荧光素在527 nm处发射(绿色)。细菌相关的β-内酰胺酶切割β-内酰胺环会导致FRET丢失并直接释放羟基香豆素(蓝色)(Jones and Padilla-Parra,2016)。所述CCF4-AM FRET报告基因分析是通用的和已被用于各种不同的应用,苏CH如跟踪分子融合(Jones和帕迪拉-帕拉,2016;谢赫,等人。,2019),生物货物的跟踪输送š至细胞溶胶(石等人。,2018),并监控translocati上的细菌细胞内(夏邦杰奥斯瓦德,20 0 4 )。由于分枝海鱼还具有β内酰胺酶(Flores的ê吨人。,2005 ),西蒙尼和他的同事开发的FRET测定来追踪的胞质易位结核分枝杆菌在THP-1细胞(西蒙尼等人,2012 )。最近,我们将这种方法用于调查EsxAB在海分枝杆菌胞质转运中的作用(Acosta等,2014; Zhang等,2016; Aguilera等,2020)。RAW264.7细胞用M.海鱼和结果被一个下获得荧光计,提供一个QUIC K,定量方法对结果进行分析。此外,由于海洋分枝杆菌是BSL-2病原体,并且在其ESX-1基因座中具有相似性(Tobin和Ramakrishnan ,2008),因此它为研究EsxAB复合物提供了一种安全,直接的方法。在这里,我们描述了我们的程序直接监控的胞浆易位分枝海鱼在RAW264.7通过CCF4-AM FRET检测使用细胞荧光。

关键字:CCF4-AM, 海洋分支杆菌, EsxA, EsxB, 荧光共振能量转移



材料和试剂



接种环(BD,Ç atalog号:220216)
刮板机(Fisherbrand ,目录号08-100-241)
T - 75烧瓶(Thermo Scientific,目录号:12-566-440)
50 ml离心管(Corning,目录号:352098)
6孔板(Corning,目录号:3516)
10 ml注射器(Fisherbrand ,目录号14-955-459)
22号针(BD,目录号305900)
27号针(BD,目录号305109)
5 µm过滤器(Millex ,目录号:SLSV025LS)
巴斯德移液器(Fisherbrand ,目录号13-678-6b)
铝箔(Fisherbrand ,目录号01-213-102)
薄壁试管(Fireflysci ,目录号:30FL)
435 nm长通滤光片(Andover Corporation,目录号:435FG03-50S)
7H9培养基(Difco公司,Ç atalog号:271310)
OADC富集(BD,ç atalog号:211886)
Tween-80 (Sigma-Aldrich,目录号:P1754)
卡那霉素单硫酸盐(金生物,Ç atalog号:K-120-5)
的DMEM培养基(g ^ IBCO ,Ç atalog号:10566-016)
胎牛血清(ģ IBCO ,Ç atalog号:A4766801)
青霉素和链霉素(ģ IBCO ,Ç atalog号:15070063)
磷酸盐缓冲盐水(Sigma-Aldrich公司,Ç atalog号:P3813)
CCF4 -AM FRET加载解决方案(生命牛逼echnologies,Ç atalog号:K1085)
注意:此套件包含该过程中所述的解决方案A,解决方案B和解决方案C。

DMSO (Sigma-Aldrich,目录号:D8418)
4%多聚甲醛
的NaCl (FISHERBRAND ,Ç atalog号:18-606-420)
氯化镁(FISHERBRAND ,Ç atalog号:18-604-047)
的CaCl 2 (FISHERBRAND ,Ç atalog号码:AA4428030)
氯化钾(频谱化学,Ç atalog号:18-605-507)
葡萄糖(Sigma-Aldrich,目录号:G7021)
4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N' - (2-乙磺酸(HEPES) )(费希尔乙ioreagents ,Ç atalog号:BP310)
三(费舍尔生物试剂,Ç atalog号:BP152-500)
EM媒体(请参阅食谱)






设备



生物安全柜(Labconco )
涡流(VWR,Ç atalog号:58816-121)
血球(豪塞尔科学,Ç atalog编号:S17040)
倒置显微镜(VWR,Ç atalog号:Vistavision )
细胞成像系统(生命Ť echnologies,Ç atalog号:4471136)
分光光度计(Amersham Biosciences,型号:U ltrospec 10)
台式离心机仪(Eppendorf,ç atalog号:5810R )
CO 2孵化器(VWR,ç atalog号3078)
荧光计(ISS,Ç atalog号:K2)
培养摇床(VWR,Ç atalog号:1585)
热板/搅拌器(赛默飞世科学,Ç atalog号:SP131325)
pH计(梅特勒托莱多,Ç atalog号:30266626)


程序



生长M. Marinu中号的细菌培养
大致感染前5 d测定,制备了M.海鱼培养感染。
接种M.海鱼入50ml 7H9培养基补充有10%OADC富集和适当抗生素根据需要,我们经常使用30毫克/毫升卡那霉素。
这种细菌培养将被用来启动一个新的细菌培养和传代,以减少细胞团ING 。
如果该细菌培养包含太多丛ED细胞,然后添加在吐温-80的浓度0.05%(V / V)ç一个被加入到所述7H9介质。
让细菌培养物在30°C下生长,同时以150 rpm的速度摇动,直到达到大约OD 600 1.2。
一旦细菌培养物生长超过对数中期,将4 ml培养物转移到46 ml补充有10%OADC的新鲜7H9培养基中。
在30°C下孵育,同时以150 rpm摇动;让细菌培养物再次生长到对数中期,大约2 d。


的培养RAW264.7小鼠巨噬细胞
感染前大约2-3天,在T-75烧瓶中用10 ml的DMEM培养基(含10%胎牛血清(FBS)和青霉素和链霉素)以100单位/ ml的浓度培养RAW264.7细胞。
让细胞在5%CO 2下于37°C增长至80-90%融合。
当天感染前,用细胞刮刀解除所有单元(在80-90%conflucence从)所述的TC烧瓶的底部。
将细胞和培养基转移到50 ml离心管中。
在室温下以160 × g离心10分钟。
除去上清液并丢弃。
ř esuspend粒料我Ñ 10毫升含有10个%FBS的DMEM培养基中的青霉素和链霉素。
使用您喜欢的任何一种方法对细胞进行计数。记录样本中细胞的近似数量。
填充6 -孔板用2ml的培养基,每孔,然后添加RAW264.7细胞接种总共2.5 × 10 6个细胞/孔。
将细胞在5%CO 2和37°C下孵育过夜。


细菌单细胞制剂
粒料向下细菌培养从部分A通过在3离心,220 ×克15分钟。
除去上清液,并将细菌重悬于50 ml PBS中。
再用PBS洗涤细胞2次,共3次。
在后第三洗涤,除去该上清,重悬沉淀10毫升的PBS。
使用注射器吸走PBS中的细菌,并通过22号针头。
注:牛逼他的步骤是可选的,但将有利于合格的文化通过一个过滤器。

用注射器吸除细菌,然后通过27号针头。
吸出细菌并通过5 µm过滤器。
用OD 600测量细菌培养物的密度。
计算要添加到的巨噬细胞取决于所需的细菌细胞的体积的感染复数(MOI)。
如果需要,通过沉淀沉淀细菌细胞并将其重悬成较小的体积。


巨噬细胞感染
在显微镜下检查RAW264.7细胞;它们应达到60%的汇合度。
吸出DMEM介质并替换为EM介质。
传染的巨噬细胞与结核分枝海鱼在10 MOI ,或在所述期望的MOI。
在5%CO 2下于30°C孵育2 h 。
之后incubati上,吸媒体。
用3 ml PBS轻轻洗涤并搅拌板。
去除PBS,再洗涤2次以去除细胞外细菌。
加入2.5 ml含10%FBS的预热EM培养基,孵育48小时。


CCF4-AM FRET检测
加载RAW264.7细胞CCF4 -AM负载解决方案由以下的制造商的建议:
载有CCF4-AM的未感染RAW264.7细胞用作阴性对照。
在RAW264.7细胞而不CCF4 -AM作为诺特尔阴性对照。
甲Chiense仓鼠卵巢(CHO)细胞系表达β内酰胺酶(CHO-β-LAC)是用来作为用于CCF4的裂解阳性对照-AM (张等人。,2016) 。
由制造商制备溶液B (100 mg / ml DMSO和0.1%乙酸中的Pluronic-F127表面活性剂)和溶液C (24%w / w PEG 400、18%TR-40 v / v在水中)。
加入182 µl DMSO溶解200 µg CCF4-AM,轻轻上下吸移。
注意:这将是制造商指定的解决方案A。

将溶液倒入20 µl等分试样中。
注意小号:

如果要储存,请在真空下干燥,并在-20 °C避光的条件下储存。
如果真空不可用,该溶液毫安ÿ被储存在-20 ℃下在该暗。该解决方案最多可稳定三个月。
加入3微升的小号olution A至30微升的小号olution B和涡流。
添加467微升的小号olution C和涡流,以产生6 × CCF4-AM衬底装载溶液。
注意:该溶液在室温下稳定12小时。

将500 µl 6 × CCF4-AM上样溶液加到100 µl细胞培养物中,制成1 × CCF4-AM上样溶液。
在避光的情况下在室温下孵育2小时。
注意:如果细胞受到干扰,则2小时的孵育将使它们有足够的时间沉降到板的底部。

吸出介质和CCF4-AM底物。
用3 ml PBS重悬细胞,并轻轻搅动板。
除去PBS,再用PBS洗涤2次,共3次。
在避光的情况下,在室温(RT)下用新鲜的4%多聚甲醛(PFA)固定细胞30分钟。
再用PBS洗涤一次,在这一阶段,细胞已准备好进行测量。


使用荧光计进行测量
轻轻重悬吨他Ç在PBS厄尔并传送它们到一个薄壁编UV比色皿。
该荧光计程序应该被设置成在405nm处激发和记录Ë之间430nm至600nm的任务,以获得所述曲线包含两个蓝色和绿色的峰(图1)。
注意:在荧光计,吨WO偏振器被放置彼此垂直的上激发光束路径和一个435纳米长通滤波器被放置在上emssition光束路径重新达斯背景和散射光。





图1 。通过FRET测量记录样品的发射光谱。一。在405nm激发后纳米,羟基香豆素将能量转移到荧光素导致在绿色发射在527纳米。含有β-内酰胺酶的细菌将逃脱吞噬体,并与细胞溶胶中的FRET染料接触,从而导致FRET的损失和在450 nm处的蓝色发射。乙。第一个峰是羟基香豆素的发射(蓝色),第二个峰是荧光素的发射(绿色)。



所述intensitiy在450纳米(蓝),我1 ,和在强度527纳米,我2 ,被测量为20迭代秒。的蓝/绿比计算为我1 /我2 (图2 )。




图2.通过将I 1除以I 2计算出足够数据的蓝/绿比。在20次迭代中测量了450 nm(蓝色)和527 nm(绿色)的强度。计算蓝/绿比率。如对海分枝杆菌观察到的较高比率,约为0.8表示信号发生了良好的变化,表明胞质易位。大号奥尔比(< 0.2)是由细菌无法逃脱吞噬体或阴性对照获得。 



数据一nalysis



的蓝/绿比分别进行平均为20个ITER从3次重复,共60次重复ations。
然后,将平均蓝色/绿色比率绘制为条形图,其中的误差线表示标准偏差,并测试其统计显着性(图3)。




图3.样品数据为不同菌株的蓝/绿比条形图。通过在条形图中绘制数据,可以看到样品之间FRET的损失。在这种情况下,野生型M.海鱼逃逸吞噬体和相互作用以导致更高的FRET染料蓝/绿比值。所述M.海鱼ΔEsxA / B是无法逃避吞噬体与导致颜色从绿色到蓝色无变化的FRET染料不相互作用。

                                                     

使用Shapiro-Wilk检验测试这些值的正态性。
如果数据未能与正常值不同,则使用单向方差分析对其进行显着性检验,否则执行Tukey-Kramer检验来检验其统计显着性。


菜谱



EM媒体(1升)
在烧杯中加入7.02克邻˚F氯化钠,0.52克的KCl ,0.26克CaCl的2 ,0.076mmol克氯化镁,0.9克葡萄糖和HEPES的6.5至水
将pH调至7.3
加水至1升


致谢



这项研究得到了NIGMS(SC1GM095475授予J. Sun),国家研究资源中心(5G12RR008124)和国家少数民族健康与健康差异研究所(G12MD007592)的资助。哈维尔·阿奎莱拉(Javier Aguilera)通过美国国立卫生研究院(RISE)为研究生提供了RISE计划,得到了美国国立卫生研究院(R25GM069621至R. Aguilera)的支持。内容仅由作者承担,并不一定代表美国国立卫生研究院的正式观点。



竞争利益



作者没有竞争利益要报告。



参考



Acosta,Y.,Zhang,Q.,Rahaman ,A.,Ouellet,H.,Xiao,C.,Sun,J.,and Li,C.(2014年)。用双光子荧光共振能量转移显微镜对分枝杆菌的胞质移位进行成像。Biomed O pt E xpress 5(11):3990-4001。
Aguilera,J.,Karki ,CB,Li,L.,Reyes,SV,Estevao ,I.,Grajeda ,BI,Qi Zhang,Arico,CD,Ouellet,H.和Sun,J.(2020年)。分枝杆菌胞质易位和毒力需要毒力因子EsxA的Nα-乙酰化。Ĵ生物学化学295(17) :5785-5794。  
Cavrois ,M.,de Noronha,C.和Greene,WC(2002)。一种灵敏且基于酶的特异性检测方法,可检测原代T淋巴细胞中的HIV-1病毒体融合。Nat B iotechnol 20(11):1151-1154 。
Charpentier ,X。和Oswald,E。(2004)。使用TEM-1β-内酰胺酶作为新的基于荧光的报告基因,鉴定肠致病性和肠出血性大肠杆菌效应物Cif的分泌和转运结构域。Ĵ乙acteriol 186(16) :5486-5495。
Flores,AR,Parsons,LM和Pavelka Jr,MS(2005)。结核分枝杆菌和耻垢分枝杆菌的β-内酰胺酶的遗传分析及对β-内酰胺抗生素的敏感性。微生物学151(2):521-532。
DM的Jones和S.的Padilla-Parra(2016)。β-内酰胺酶测定:利用FRET生物传感器分析病毒融合机制。传感器16(7):950。
Simeone ,R.,Bobard ,A.,Lippmann,J.,Bitter,W.,Majlessi ,L.,Brosch ,R.和Enninga ,J.(2012)。结核分枝杆菌引起的噬菌体破裂会导致毒性和宿主细胞死亡。PLoS Pathog 8(2):e1002507。
Shaikh,F.,Zhao,Y.,Alvarez,L.,Iliopoulou ,M.,Lohans ,C.,Schofield,CJ,Parra,SP,Siu,SWI,Fry,EE和Ren,J.S . (2019)。基于结构的,在硅片从传统的筛选识别一个强有力的埃博拉病毒抑制剂,中国医学图书馆。Ĵ中号编Ç下摆62(6) :2928至2937年。
石,SR,海因里希,T.,Juraja ,SM,Satiaputra ,JN,霍尔,CM,Anastasas ,M.,米尔斯,DM ,张伯伦,CA,温斯洛,S。和普瑞巴什,K. (2018)。β-内酰胺酶工具,用于建立细胞穿透肽的细胞内在化和胞质传递。生物分子8(3):51。   
托宾(DM)和拉玛克里希南(Lamakrishnan,L.)(2008)。海洋结核分枝杆菌与结核分枝杆菌的比较发病机制。 细胞中号icrobiol 10(5) :一○二七年至1039年。
Zhang,Q.,Wang,D.,Jiang,G.,Liu,W.,Deng,Q.,Li,X.,Qian,W.,Ouellet,H.和Sun,J.(2016)。EsxA膜透化活性在分枝杆菌胞质易位和毒力的关键作用:在谷氨酰胺5.单残基突变的影响科学代表32618:6。
Zlokarnik ,G. ,Negulescu ,PA,Knapp,TE,Mere,L.,Burres ,N.,Feng,L.,Whitney,M.,Roemer,K。和Tsien ,RY(1998)。以β-内酰胺酶为报告基因的单个活细胞的转录定量和克隆选择。科学279(5347):84-88。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 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. Aguilera, J. and Sun, J. (2021). Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay. Bio-protocol 11(8): e3991. DOI: 10.21769/BioProtoc.3991.
  2. Aguilera, J., Karki, C. B., Li, L., Reyes, S. V., Estevao, I., Grajeda, B. I., Qi Zhang, Arico, C. D., Ouellet, H. and Sun, J. (2020). Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence. J Biol Chem 295(17): 5785-5794.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。