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Phagolysosomal Trafficking Assay
吞噬溶酶体的胞内转运分析   

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Abstract

Phagolysosomal trafficking is an important innate defense pathway that clears microbes by delivering them to lysosomes, the degradative compartment of the cell. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, subverts this host defense mechanism by arresting maturation of the phagosome. The ability of Mtb to arrest its delivery to the lysosome can be demonstrated by the prolonged co-localization of bacteria containing phagosomes/vacuole with early phagosomal markers [such as, Ras- related proteins in the brain 5 (Rab5) and Transferrin receptor (TfR)], and a failure to acquire late phagosomal and lysosomal markers (such as Rab7 and LAMP1) (Deretic and Fratti, 1999, Mehra et al., 2013). Here, a protocol is outlined for infection of macrophages with mycobacterial species like pathogenic Mtb, vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) and rapidly dividing non-pathogenic Mycobacterium smegmatis (Msmeg), followed by indirect-immunofluorescence microscopy to visualize host vacuolar markers. Thereafter, automated quantification of degree of co-localization between mycobacteria and host vacuolar markers like TfR and LAMP1 is done by processing the binary images of bacteria using mathematical tools. This results in quantification of the mean fluorescence intensity (MFI) of these host markers directly around the bacteria/bacterial clusters with increased sensitivity relative to when done manually. By manipulating host or pathogen, this assay can be used to evaluate host or bacterial determinants of intracellular trafficking. The basic method can be applied to studying trafficking of other bacteria or particles like beads, although the kinetics of infection and phagosome maturation will depend upon the phagocytic cargo. The mathematical analysis tools are available in many standard imaging analysis programs. However, any adaption for similar analysis should be confirmed by the individual user with their imaging and analysis platform.

Keywords: Macrophage infections(巨噬细胞的感染), Vacuolar staining(液泡染色), Trafficking of bacteria(细菌人口)), Phagosomal fluorescence analysis(吞噬体荧光分析), Morphology and boolean logic operators(形态和布尔逻辑算子)

Materials and Reagents

Note: All work with live Mtb must be performed in a Biosafety Level 3 (BSL3) facility according to institutional standards of practice.

  1. Macrophages, either primary macrophages, such as C57BL/6 bone marrow-derived macrophages (BMDMs) or a macrophage cell line (such as RAW264.7) 
    Note: BMDMs can be isolated as described (Banaiee et al. 2006; Nagabhushanam et el., 2013). RAW264.7 cells can be purchased from ATCC (ATCC, catalog number: TIB-71 ).
  2. L929 cells (ATCC, catalog number: CCL-1 )
  3. Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco®, catalog number: 11965 )
  4. Fetal Bovine Serum (FBS) (heat inactivated) (Life Technologies, Gibco®, catalog number: 10082147 )
  5. 1 M HEPES solution (Life Technologies, Gibco®, catalog number: 15630-056 )
  6. 200 mM L-glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
  7. Penicillin-Streptomycin solution (10,000 U/ml) (Life Technologies, Gibco®, catalog number: 15140-122 )
  8. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 10010-023 )
  9. Eight well Permanox chamber slide (Thermo Fisher Scientific, Nunc Lab-Tek Chamber Slides, catalog number: 177445 )
  10. Eight well chamber coverglass (Thermo Fisher Scientific, Nunc Lab-Tek Chamber coverglass, catalog number: 155411 )
  11. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  12. Bovine serum albumin (BSA) (fraction V) (Thermo Fisher Scientific, catalog number: BP1600 )
  13. Detergents: Saponin (Sigma-Aldrich, catalog number: 47036 ), Triton- X100 (Sigma-Aldrich, catalog number: X100) and/or Tween-20 (Thermo Fisher Scientific, catalog number: BP337 )
  14. Primary antibodies to detect host cellular markers 
    For example, recycling endosomes and early phagosomes can be labeled with mouse anti-transferrin receptor (anti-TfR) antibody (Life Technologies, InvitrogenTM, catalog number: 136800 ); Late endosomes and lysosomes stain with rabbit anti-LAMP1 antibody (Abcam, catalog number: 24170 ). 
    Notes: 
    1. If Mtb infected slides are to be removed from the BSL3 for imaging, the antibodies chosen need to work after fixation cum sterilization methods like long fixation as mentioned below in step A11, note a. Some of the commercially available antibodies may loose recognition or weakly recognize their epitopes after long fixation. 
    2. It is critical that polyclonal antibodies were not raised in animals given Freund’s adjuvant, as then they will directly recognize Mtb in addition to whatever cellular marker they were raised against. All antibodies should be tested to verify that they do not directly recognize Mtb. 
  15. Secondary antibodies for immunofluorescence
    Secondary antibodies are available against different species and in different colors and user may choose depending on the primary antibodies being used. They are adsorbed against multiple species to minimize species cross reactivity during immunostaining. For example, Goat anti- mouse Alexa 594 (Life Technologies, Molecular Probes®, catalog number: A11032) and Goat anti-rabbit Alexa 594 (Life Technologies, Molecular Probes®, catalog number: A11037 ).
     Of note, Mtb exhibits autofluorescence, with an emission maximum at 475 nm when excited at 405 nm, and thus are visualized by many DAPI filters (Patiño et al., 2008). Therefore, secondary antibodies should be chosen that do not fluoresce in this range. 
  16. Lysotracker Red DND-99 (1 mM stock in DMSO) (Life Technologies, Molecular Probes®, catalog number: L-7528 )
    Note: Lysotracker dyes are available in different colors and one may choose depending on the color requirement. 
  17. Dextran (TexasRed, 10,000 MW, Lysine fixable) (Life Technologies, Molecular Probes®, catalog number: D-1863 ) (make 25 mg/ml stock in PBS, stored in dark in -20 °C)
    Note: Dextran is available in different colors and molecular weights and again one may choose depending on requirement and desired goals of the experiment.
  18. Vectashield mounting media (Vector Laboratories, catalog number: H-1000 )
  19. Middle brook 7H9 broth (Difco, catalog number: 271310 )
  20. Albumin-dextrose-catalase (ADC) (BD, catalog number: 212352 )
  21. Oleic-albumin-dextrose-catalase (OADC) (BD, catalog number: 212351 )
  22. Nail polish (clear)
  23. Immersion oil (Microscope 50CC Immersion oil) (e.g. Nikon Corporation, catalog number: IB-MA-MXA20234 )
  24. 4% paraformaldehyde solution in PBS (see Recipes)
  25. DMEM complete media (see Recipes)
  26. DMEM/L929 complete media (see Recipes)
  27. L-Cell conditioned media (see Recipes)
  28. 2% BSA in PBS containing 0.1% saponin (see Recipes)
  29. 2% BSA in PBS containing 0.1% triton X-100 (see Recipes)
  30. 7H9 complete media (see Recipes)
  31. Fixative (see Recipes)
  32. Blocking solution (see Recipes)

Equipment

  1. Spectrophotometer (measure the OD600 i.e. optical density at wavelength of 600 nm of the mycobacterial cultures using cuvettes)
  2. Disposable 1.5 ml cuvettes (Perfector Scientific, catalog number: 9003 )
  3. Disposable sterile filter system (500 ml, 0.22 µm pore size) (Corning, catalog number: 430758 )
  4. 30 ml square media bottles (Thermo Fisher Scientific, Nalgene, catalog number: NE/2019-0030 )
  5. 50 ml, 15 ml falcon tubes (with plug seal caps) (Corning, catalog numbers: 430052 and 430290 )
  6. Coverslip (22 x 50 mm, thickness#1, 0.13-0.17 mm) (Thermo Fisher Scientific, catalog number: 12-545C )
  7. Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Beckman Coulter, model: Allegra X-15R ; bench top centrifuge with SX4750 rotor)
    Note: Mtb cultures should be handled in BSL3 facility according to institutional standards of practice.
  8. 37 °C shaker incubator with aerosol containment units for Mtb liquid cultures
  9. Beckman aerosolve canisters for centrifuging mycobacterial cultures in falcon tubes (e.g. Beckman Coulter, catalog number: BK359232 )
  10. Multiwell-Plate Carrier Covers (e.g. Beckman Coulter, more on this link https://www.beckmancoulter.com/wsrportal/techdocs?docname=GX-TB-012)
  11. 37 °C shaker incubator with aerosol containment units for Mtb liquid cultures
  12. Epifluorescence microscope [e.g. Nikon Eclipse TiE/B model equipped with 60x; Plan-Apochromat, NA 1.4 oil immersion objective, Ti Z drive, high resolution monochrome charge-coupled device (CCD) digital camera; Photometric Cool SNAP HQ2 and appropriate filter sets for DAPI, FITC and TexasRed channel]

Software

  1. Nikon Imaging Software-Elements Advanced Research (NIS-Elements AR) version 3.2 software with deconvolution module
  2. Graph Pad Prism software

Procedure

  1. Infection of macrophages
    Notes:
    1. All work with live Mtb to setup infection (steps A1 and A3-11 below) should be done in bio-safety level 3 (BSL3) facility.
    2. BCG and Msmeg cultures should be handled outside BSL3 facility but in biosafety class II cabinets or as per institutional standards of practice. 
    3. Centrifugation of Mtb and BCG cultures (steps A4-6 below) and infected samples (step A9 below) should be done using suitable aerosol canisters and multiwell plate cover or as per institutional standards of practice. These canisters and multiwell plate carriers with covers should be opened for loading and unloading of Mtb/BCG culture containing falcons or infected plates in the bio-safety class II cabinets during centrifugation.
    1. Starting with frozen bacterial stocks (prepared from mid-log phase culture i.e. culture with OD600 between 0.5 to 1.0 units frozen in 18% glycerol at -80 °C) inoculate liquid cultures of mycobacteria in 10 ml of 7H9 complete media in 30 ml square media bottles. Include antibiotics as appropriate (for example, to select plasmids containing GFP with kanamycin as the selection marker, include kanamycin at 5 µg/ml). Incubate at 37 °C with shaking at 90-110 rpm in aerosol containment units. Mtb and BCG double approximately every 20 h in 7H9 complete media at 37 °C so the cultures will take few (~4-5) days to reach mid-log phase. The cultures maybe diluted if required into fresh media (e.g. if antibiotic used is prone to degradation during culture) at appropriate intervals. Msmeg doubles approximately every 3 h and so dilute an overnight grown culture in the morning to reach mid-log phase during the day to use for infection. The timing will depend upon the particular strain, conditions, and starting inoculum.
    2. Plating of macrophages should be done one day prior to infection in bio-safety class II cabinet in the tissue culture lab. Plate the cells in 250 µl of either DMEM or DMEM/L929 complete media per well in 8 well chamber slide. BMDMs can be plated at a density of 1 x 105 per well. If RAW264.6 macrophage cell line is used, it can be seeded with a density of 6 x 104 per well in DMEM complete media. Incubate the cells in 37 °C incubator with 5% CO2 atmosphere. 
      On the day of Mtb infection, transfer the slides to the 37 °C incubator with 5% CO2 atmosphere in the BSL3 facility before proceeding further.
      Of note, coverslips placed in 24 well plate can also be used for plating the macrophages for infection. However, 8 well chamber slide and cover glass offer sterile and RNase free plating conditions with minimal use of reagents. In addition, multiple conditions can be tested with minimal well to well variation like different host markers can be knocked down prior to infection to see the role of host protein in phagolysosomal trafficking or multiple bacterial strains can be infected in different wells of the same slide.
    3. Measure the OD600 of 1ml of the culture in cuvette using spectrophotometer. Dilute the culture if required so as to have the OD600 of the culture between 0.5 to 1.0 OD600 on the day of infection of macrophages.
    4. Transfer culture into 15 ml falcon tube and spin at 1,500 x g for 5 min at room temperature. 
    5. Remove the supernatant and resuspend the pellet in 5 ml of PBS and again spin at 1,500 x g for 5 min at room temperature. Repeat it once more to remove carried over Tween 80 from 7H9 complete media.
    6. Resuspend the pellet in 5 ml of DMEM complete media for RAW 264.7 macrophages and DMEM/L929 complete media for BMDM and transfer it to 50 ml falcon. Spin at low speed of 450 x g for 5 min at room temperature to remove bacterial clumps by pelleting. Transfer the supernatant carefully avoiding the pellet to a fresh 15 ml falcon tube. The supernatant is devoid of mycobacterial clumps with substantial single cell population and is therefore used to infect macrophages.  
      Note: It is not unusual to remove ~ 90% of the mycobacteria by pelleting in this step. For example, if at step A3, the OD600 of a 10 ml culture of Mtb is 0.5 then expect the supernatant after low speed spin to have an OD600 ~ 0.05 to 0.1 depending on the degree of clumping. 
    7. Determine the OD600 of the supernatant harvested in step A6. Convert the OD600 to number of bacteria. This value will vary depending upon the shape, size and internal light absorbing components of the bacteria and may be distinct for different strains of the same species. One should predetermine the conversion factor. For example, a conversion factor such as OD600 of 1.0 = 500 x 106 colony forming units (CFU)/ml can be determined as below: 
      1. a. A starting culture of “x” OD600 is serially diluted and a volume of each dilution is plated to give CFUs.
      2. The dilution which give colonies in countable range and the number of colonies for that dilution are used to calculate “y” CFU/ ml using the formula:
        CFU/ml = (# colonies) * (dilution factor)/ (volume plated in ml). 
      3. This gives x OD600 = y CFU/ml. Now OD600 of 1 = y/x CFU/ml.
    8. Calculate the volume of the bacterial suspension prepared in step A6 that is required to infect macrophages at a multiplicity of infection (MOI) of ~ 1-10. Add this volume to each the well. If necessary, make a dilution so that at least 10-50 µl is added to each well in a total volume of 200 µl. Several MOIs should be tested and the optimal MOI will depend upon details of the experimental conditions. 
    9. Spin the slide at 50 x g for 2 min at room temperature to synchronize the infection using multiwell plate carriers with covers in the swing bucket rotor. Incubate in 37 °C incubator with 5% CO2 atmosphere for 3 h. Different strains may differ in infectivity of the macrophages.
    10. After 3 h, remove uninternalized bacteria by washing three times with 300 µl pre-warmed PBS. Add 300 µl of DMEM or DMEM/L929 (for RAW cells or BMDMs, respectively) and incubate in 37 °C incubator with 5% CO2 atmosphere. It is possible to perform this step after a shorter time period, although bacterial uptake will be lower.
    11. At desired time points [such as 3 h post-infection (hpi), 12 hpi, and 24 hpi], remove the media. Fix in 1% PFA/PBS at 4 °C overnight for Mtb infected samples or as per institutes bio-safety guidelines before removing them from the BSL3. Fix in 4% PFA/PBS at room temperature, 15 min, for Msmeg and BCG infected samples. 
      Notes:
      1. As per biosafety rules, Mtb infected slides should be sterilized before taking them out of BSL3 for immunostaining and imaging (Schwebach et al., 2001). This can be achieved by long fixation method; fixing in 1% PFA/PBS, overnight (minimum of 12 h) at 4 °C or as per institutional’s standards of practice. 
      2. For alternate fixatives, like methanol or acetone, the dishes for plating macrophages and infection should be compatible with organic solvents.

  2. Staining for immunofluorescence microscopy
    For immunofluorescence (IF) microscopy, one has to pre-optimize the conditions for immunostaining which includes fixatives (see step A11, notes a-b), detergent to permeablize the cells (triton X-100, saponin or tween-20), blocking and dilution for each primary antibody before performing co-localization experiments (Goldenthal et al., 1985). During standardization, the specificity of immunostaining can be confirmed by silencing the host marker by RNA interference (RNAi). It is important to include controls that would help in setting acquisition exposures and also serve as a control for non-specific staining during fluorescence microscopy as explained further in step C21. For Mtb infected samples, primary antibodies for a specific host marker should be screened for recognition of specific epitopes for optimal sensitivity in macrophages which have been fixed for long to sterilize Mtb which is in contrast to BCG and Msmeg infected samples which are fixed for short (as described in step A11 of the protocol).
    1. Wash out the fixative well with 300 µl of PBS three times. 
    2. Blocking: Incubate in blocking buffer for 1 h at room temperature. For example, blocking buffer can be 2% BSA in PBS containing 0.1% detergent optimized for the primary antibody. The detergent permeablizes the plasma membrane so that the antibody can enter the cell, and BSA blocks to prevent non-specific staining.
      For the commercially available primary antibodies mentioned in materials and reagents, transferrin receptor (TfR) and LAMP1, add 200 µl of 2% BSA in PBS-0.1% triton X-100 and 2% BSA in PBS-0.1% saponin, respectively, to the corresponding wells to block.
    3. Primary antibody: Add 150 µl of the diluted primary antibody to the well and incubate overnight at 4 °C. Dilute the primary antibody in the 2% BSA in PBS-0.1% detergent as standardized. For the primary antibodies mentioned in materials and methods, dilute TfR (1: 250 v/v) and LAMP1 (1: 1,000 v/v) in 2% BSA in PBS-0.1% triton X-100 and 2% BSA in PBS-0.1% saponin, respectively. 
    4. Wash with PBS-0.1% detergent three times, each time for 5 min at room temperature to remove excess and non-specifically bound antibody e.g. use PBS -0.1 % triton X-100 for transferrin receptor and PBS -0.1 % saponin for LAMP1 antibodies used in step B14.
    5. Secondary antibody: Depending on species in which primary antibody was raised, use the appropriate anti-species (mouse or rabbit) secondary antibody labeled with appropriate fluorophore. Add 150 µl of the diluted secondary antibody and incubate for 1 h at room temperature in the dark to prevent bleaching of the fluorophore. Dilute the secondary antibody 1: 250 in 2% BSA in PBS-0.1% detergent e.g. use anti-mouse Alexa 594 in 2% BSA in PBS-0.1% triton X-100 to detect anti-TfR and dilute anti-rabbit Alexa 594 in 2% BSA in PBS-0.1% saponin to detect anti-LAMP1.
    6. Repeat step B15. Protect from light to prevent photo-bleaching of the secondary antibody.
    7. Remove the chambers from the top of the slide by gentle upward pressure and peel off the rubber gasket (see Figure 1).
    8. Put ~2 µl of anti-fade in the center of each of the eight well on the slide. Mount a clean rectangular coverslip and seal the sides of the coverslip with nail polish.
    9. Although not described in detail here, alternatively, it is also possible to do live cell staining of lysosomes using lysotracker dyes or loading them with fluorescent dextran (e.g. TexasRed-dextran). The kinetics of live staining has mainly two variables, concentration and time and depending on the desired goals one may best design the experiment to couple it with infection. A brief understanding towards it usage is provided: 
      1. Lysotracker is a cell permeable, acidotrophic stain that can be used to label lysosomes in macrophages without the need for fixation. Staining conditions vary and users may standardize concentration of the dye and time of staining (30 min to 2 h) depending on the actual experiment. 
        1. Plate the macrophages and infect them as described in section A. At the last 30 min of the desired time point post-infection of macrophages, remove media from well, add 150 µl of 200 nM lysotracker per well and incubate at 37 °C. Protect the samples from light hereafter to prevent photobleaching. Turn off the light in the biosafety cabinet while working with the stained sample to prevent photo-bleaching.
        2. Wash the well three times with PBS to remove excess of the lysotracker.   
        3. Add PBS and quickly proceed for image acquisition with the fluorescence microscope. Further incubation in dye free media often leads to fading of the signal and cell blebbing. 
          Note: Msmeg and BCG infected macrophage samples can be directly taken for live image acquisition and so macrophages in this case should be plated on 8 well chamber cover glass instead of the slide so that the chambers do not have to be removed. In case of Mtb infected macrophages, after live labeling with lysotracker, the samples should be fixed as per institutes bio-safety guidelines before taking them out of BSL3 for imaging. They can then be washed with PBS to remove fixative and mounted with the anti-fade.
      2. TexasRed-dextran (10 kD) is freely permeable to the endocytic and lysosomal vesicular network of the cell and so can be used to label lysosomes before infection by pre-loading the macrophages: 
        1. Plate the macrophages as in step A2. Remove the plating media from the well and treat with 150 µl of 1 mg/ml solution of dextran (diluted in PBS) for 1h at 37 °C for cellular uptake by fluid phase endocytosis.
        2. Wash with pre-warm complete DMEM or DMEM/L929 complete media (as required) twice, observe under the microscope and if required wash more with pre-warmed PBS twice. 
        3. Add back warm complete DMEM or DMEM/L929 complete media (as required) and incubate for 4 h in 37 °C incubator with 5% CO2 to chase the TexasRed-dextran to lysosomes. This chase period can be done overnight followed by infection the next day.
        4. Infect the macrophages and fix the samples at the desired time points post-infection.

  3. Image acquisition
    Note: Appropriate training should be acquired before operating any fluorescence or confocal microscope for image acquisition as per individual or core facility guidelines.  
    1. For image acquisition (see Reference 8 for more details), it is important to have these controls: 
      1. Fixed but unstained as a control for background autofluoresence of the cells and bacteria.
      2. Secondary alone control to check for non-specific staining.
      3. Single color controls in case of multi-color labeling experiment e.g. when doing dual immunolabeling of two different markers with compatible staining procedures in the same well. This is essential to collect images for each single color in all channels at exactly same settings as being used to acquire image of the multi-color sample. It helps to correct for the crosstalk (excitation for one dye with incident light indented for the other dye) and bleed-through (emission of one dye into detector for other dye). 
      4. If single color controls indicate bleed through, it is best to do immunolabeling of only one marker in each well.
    2. Images are acquired using the instructions specific to the fluorescence or confocal microscope at high magnification e.g. with 60x 1.4 NA oil immersion objective using Nikon Eclipse TiE/B microscope. Set optimal exposure levels for differential interference contrast (DIC) and required color channel (DAPI, GFP and/or TxRed) without saturating the pixel intensity. Using the auto exposure tool with single color controls is useful in estimating an exposure level to prevent cross-talk.  
    3. After phagocytosis, mycobacteria with slender rod like morphology is mainly localized in vesicular structures/phagosomes in the cytoplasm of the cell up to 48 h (van der Wel et al., 2007). Mycobacteria is 0.2 to 0.5 µM in width with average width of 0.35 µM and so appropriate z-stacks should be obtained. A minimum of three fields with 10 to 15 macrophages per field should generally be acquired per well to generate a minimum of 100 regions of interest (ROI) per condition to use for calculation of statistical significance. 
      Note: When using the autofluorescence of Mtb in DAPI channel to visualize bacteria, it is advised to keep the exposure of the sample to exciting incident light at 405 nm to minimum since it quickly bleaches the autofluorescence of Mtb. 
    4. Deconvolute the images if required as in case of acquisition with Nikon Eclipse TiE/B epifluorescence microscope and correct for background on all images before analysis.

  4. Image analysis
    Endosomal/lysosomal markers localize around the phagosomal cargo (Fratti et al., 2001; Hava et al., 2008; Delaby et al., 2012; Mottola et al., 2014). In the illustrated example in Figure 2A, the host marker, transferrin receptor (TfR) is shown in grey and the phagosomal cargo, Mtb is in green. Here the degree of co-localization of the host markers and bacteria/bacterial clusters is variable between phagosomes (see phagosomes 1, 2, 3 and 4 in Figure 2A). Qualitative analysis done visually suggests phagosomes, 2 and 3 have high and phagosomes, 1 and 4 have low TfR staining around bacteria. For quantification of the host vacuolar staining around bacteria/bacterial clusters, I describe automated analysis done using NIS-Elements AR version 3.2 using Figure 2 as an example. 
    1. Create a binary image in the color channel of the bacteria (green). In binary images, the background will have 0 pixel intensity and the selected object (bacteria) will have a pixel intensity of 1. In the given example, Figure 2B, this can be achieved by “Thresholding” the intensity in the color channel of the bacteria. Additionally, in NIS Elements Advanced Research software, the “Autodetect all” tool can be used which works by detecting the intensity of the pixels under the cursor and so the cursor movement helps to mark all the bacteria in a given field.
    2. Using the binary image of the bacteria, “Dilate” three times to enlarge the marked bacteria in the active window. Copy this image with dilated bacteria to clipboard which is a reference binary image to be used later (Figure 2C). 
    3. Now using the active window of the dilated bacterial image, “Erode” three times to shrink the enlarged area in step D26. This creates a current binary image to be used later (Figure 2D). 
    4. Use the command “eXclusive OR” (XOR) function on the two images (Figure 2E), the reference and current binary from steps D26-27, respectively to generate a processed result binary image where “rings” are marked around bacteria/bacterial clusters (Figure 2F).
    5. In the active result binary image, converting the rings to regions of interest (ROIs) (see ROI IDs: 1-4 in Figure 2G). 
    6. Open the image of the vacuolar marker (e.g. TfR) and measure the ROI in that channel to obtain the mean fluorescence intensity (MFI) for the ROIs (see Figure 2H).
      Notes: 
      1. Validation of the analysis: The visual scoring of high co-localization on phagosomes 2 and 3 and less on phagosome 1 and 4 in Figure 2A correlates with automated quantification in Figure 2H with ROI ID: 2 having maximum mean intensity and ROI ID: 1 has the least mean intensity of fluorescence.
      2. The count number of three for dilation and erosion should be optimally determined for each phagosomal cargo by individual user on set of images to best capture the signal intensity of the vacuolar marker.
    7. Plot the MFI for all the ROI on the Y-axis for each well. Graph display, statistical calculations and P values can be determined using Graph Pad Prism software.
      Notes:
      1. Morphology operators (dilate, erode) and Boolean logic operations are integrated as processing tools in many image analysis softwares for easy use. However, they are based on complex mathematical theories and so I refer to literature (Serra, 1987; Dougherty and Lotufo, 2003) to be careful with the interpretations of these processing parameters. 
      2. Individual user should optimize automated analysis of co-localization of different host markers with phagosomal cargoes and tally the results with visual scoring to validate the analysis as described above in section D of the protocol. 
      3. The results of automated analysis of a trafficking experiment (for example, co-localization of Mtb with TfR and LAMP1 at 3 and 24 hpi in Mtb infected macrophages) should be consistent with visual scoring done manually by a blinded observer.


      Figure 1. Illustration showing detachment of the top chambers from the 8 well chamber slide
       
      Remove the washing buffer from the wells (A) and insert blade as shown (B) and put gentle upward pressure which will detach the top plastic chamber (C) revealing a rubber gasket underneath (D). Peel off the rubber gasket gently (E).



         
      Figure 2.  Automated analysis of co-localization of host markers with mycobacterial phagosome. A. Immunostained image of Mtb infected RAW 264.7 macrophage at 24 hpi. A: Host marker (TfR) in grey and Mtb in green co-localize on phagosomes 1, 2, 3, 4 (pointed by blue arrows) to various degrees. Images have been pseudo-colored for illustration. B-H: Binary image processing to quantify co-localization as described in steps 25 to 30 of the section D of the protocol: Here, black, grey and yellow circles are symbolic representation of images C, D and F respectively. E shows pictorial representation of the effect of Boolean (XOR) operator on the images C and D image to yield F. H gives the quantification of mean fluorescence intensity (MFI) of TfR in the different ROIs with ID from 1 to 4.     

Recipes

  1. 4% Paraformaldehyde solution in PBS (pH 7.4) (1 L)
    Paraformaldehyde 
    40 g
    10x PBS 
    100 ml
    Deionized water
    800 ml
    Note: Paraformaldehyde fumes are toxic. All work should be done in a ventilated fume hood.
    1. Heat 800 ml of on a hot plate to 60 °C.
    2. Add 40 g of paraformaldehyde while stirring on a hot plate.
    3. Add 50 µl of 10 N of sodium hydroxide (NaOH), continue to stir.
    4. Allow solution to stir until paraformaldehyde dissolves.
    5. Add 25 ml of 10x PBS and mix.
    6. Measure the pH of the solution using appropriate pH strips.
    7. Add water to a final volume of 1 L.
    8. Filter 4% paraformaldehyde through 0.45 µm filter.
    9. Aliquot and freeze at -20 °C for long term storage.
  2. DMEM complete media 
    DMEM
    435 ml
    1 M HEPES
    10 ml
    0.2 M L-glutamine
    5 ml
    Heat inactivated fetal bovine serum 50 ml
    Filter sterilize through 0.2 µM filter and stored at 4 °C
    No antibiotics should be included in the media if it is going to be used for infections
  3. DMEM/L929 complete media
    DMEM                                         
    390 ml
    Heat inactivated FBS
    50 ml
    L929-cell conditioned media
    50 ml
    0.2 M L-glutamine
    5 ml
    0.1 M sodium pyruvate
    5 ml
    Filter sterilize through 0.2 µM filter and stored at 4 °C
    No antibiotics should be included in the media if it is going to be used for infections.
  4. Preparation of L-Cell conditioned media
    L929-cell medium
    DMEM                                                           
    440 ml
    Heat inactivated FBS
    50 ml
    200 mM L glutamine
    5 ml
    100x Non-essential amino acids
    5 ml
    1,000x β-mercaptoethanol
    0.5 ml
    1. Thaw 1 vial of L929 cells, add 1 ml of warm media to the vial and plate on one 15 cm TC dish in at least 35 ml of warm media.
    2. Allow to grow to confluence, usually takes 3-4 days when starting from frozen stock.
    3. Rinse the cells with 1x PBS and then cover with 10 ml 1x Trypsin EDTA and incubate at 37 °C for 1-5 min until the cells come off the bottom of the plate with gentle pipetting. 
    4. Add an excess of L-cell media to the cells (3x the volume of Trypsin EDTA added) and pipette up and down to break clumps.
    5. Centrifuge at 650 x g for 5 min.
    6. Resuspend the cells in 25 ml of media.
    7. Add 1 ml of cells to each of 25 of 15 cm TC dishes containing 38 ml of growth media.
    8. Allow cells to grow for about 5-7 days, the cells should reach confluence on day 6. Pipet off media and filter with a 0.22 µM filter.
    9. Aliquot into 50 ml vials and freeze at -80 °C for use up to six months.
  5. 2% BSA in PBS containing 0.1% saponin (50 ml)
    1. Weigh 0.1 g of saponin and dissolve in 100 ml of 1x PBS. Filter through 0.45 µm and stored at room temperature.
    2. Dissolve 2 g BSA in 50 ml of PBS-0.1% saponin gently (prepare fresh).
  6. 2% BSA in PBS containing 0.1% triton X-100 (50 ml)
    1. Weigh 0.1 g of triton X-100 (w/v) and dissolve in 100 ml of 1x PBS. Filter through 0.45 µm and stored at room temperature.
    2. Dissolve 2 g BSA in 50 ml of PBS-0.1% triton X-100 gently (prepare fresh).
  7. 7H9 complete media (1 L)
    7H9 powder                     
    4.7 g
    50% glycerol
    4.0 ml
    20% Tween 80
    2.5 ml 
    Water
    to 900 ml
    1. Dissolve 7H9 powder in water and add glycerol and Tween 80. 
    2. Adjust the volume of water to give a final volume of 900 ml media. 
    3. Add 100 ml of OADC for Mtb complete media or 100 ml of ADC for Msmeg and BCG complete media. 
    4. Sterilize through 0.22 µM filter and store at 4 °C.  
    5. Alternatively, autoclave after dissolving 7H9 and glycerol in about 900 ml water and then supplement with sterile solution of tween-80 and 100 ml of ADC/OADC and store at 4 °C.
  8. Fixative
    4% PFA/PBS for fixing BCG and Msmeg 
    1% PFA/PBS for fixing Mtb
  9. Blocking solution
    2% BSA in PBS containing 0.1% detergent

Acknowledgments

I thank Jennifer A. Philips for the supervision and development of this protocol. This protocol was adapted from the published work Mehra et al. (2013). The work was supported by grants and fellowships from the NIH (R01 AI087682), the Doris Duke Charitable Foundation, the Infectious Disease Society of America, the Michael Saperstein Medical Scholars Research Fund (New York University School of Medicine), Potts Memorial Foundation and the American Society of Microbiology.

References

  1. Banaiee, N., Jacobs, W. R., Jr. and Ernst, J. D. (2006). Regulation of Mycobacterium tuberculosis whiB3 in the mouse lung and macrophages. Infect Immun 74(11): 6449-6457.
  2. Delaby, C., Rondeau, C., Pouzet, C., Willemetz, A., Pilard, N., Desjardins, M. and Canonne-Hergaux, F. (2012). Subcellular localization of iron and heme metabolism related proteins at early stages of erythrophagocytosis. PLoS One 7(7): e42199.
  3. Deretic, V. and Fratti, R. A. (1999). Mycobacterium tuberculosis phagosome. Mol Microbiol 31(6): 1603-1609.
  4. Dougherty, E. R. and Lotufo, R. A. (2003). Hands-on morphological image processing. Spie Bellingham, WA.
  5. Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S. and Deretic, V. (2001). Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J Cell Biol 154(3): 631-644.
  6. Goldenthal, K. L., Hedman, K., Chen, J. W., August, J. T. and Willingham, M. C. (1985). Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins. J Histochem Cytochem 33(8): 813-820.
  7. Hava, D. L., van der Wel, N., Cohen, N., Dascher, C. C., Houben, D., León, L., Agarwal, S., Sugita, M., van Zon, M. and Kent, S. C. (2008). Evasion of peptide, but not lipid antigen presentation, through pathogen-induced dendritic cell maturation. Proc Natl Acad Sci U S A 105(32): 11281-11286.
  8. Mehra, A., Zahra, A., Thompson, V., Sirisaengtaksin, N., Wells, A., Porto, M., Koster, S., Penberthy, K., Kubota, Y., Dricot, A., Rogan, D., Vidal, M., Hill, D. E., Bean, A. J. and Philips, J. A. (2013). Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog 9(10): e1003734.
  9. Mottola, G., Boucherit, N., Trouplin, V., Oury Barry, A., Soubeyran, P., Mege, J. L. and Ghigo, E. (2014). Tropheryma whipplei, the agent of whipple's disease, affects the early to late phagosome transition and survives in a Rab5- and Rab7-positive compartment. PLoS One 9(2): e89367.
  10. Nagabhushanam, V., Solache, A., Ting, L. M., Escaron, C. J., Zhang, J. Y. and Ernst, J. D. (2003). Innate inhibition of adaptive immunity: Mycobacterium tuberculosis-induced IL-6 inhibits macrophage responses to IFN-gamma. J Immunol 171(9): 4750-4757.
  11. Patiño, S., Alamo, L., Cimino, M., Casart, Y., Bartoli, F., García, M. J. and Salazar, L. (2008). Autofluorescence of mycobacteria as a tool for detection of Mycobacterium tuberculosis. J Clinical Microbiol 46(10): 3296-3302.
  12. Schwebach, J. R., Jacobs, W. R., Jr. and Casadevall, A. (2001). Sterilization of Mycobacterium tuberculosis Erdman samples by antimicrobial fixation in a biosafety level 3 laboratory. J Clin Microbiol 39(2): 769-771.
  13. Serra, J. (1987). Morphological optics. J Microsc 145(Pt 1): 1-22.
  14. van der Wel, N., Hava, D., Houben, D., Fluitsma, D., van Zon, M., Pierson, J., Brenner, M. and Peters, P. J. (2007). M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129(7): 1287-1298.
  15. Waters, J. C. (2009). Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 185(7): 1135-1148.

简介

吞噬溶酶体运输是重要的先天防御途径,通过将其递送至溶酶体,即细胞的降解区室来清除微生物。结核病的致病因子结核分枝杆菌(Mtb)通过阻滞吞噬体的成熟来破坏这种宿主防御机制。 Mtb阻止其递送至溶酶体的能力可以通过含有吞噬体/液泡的细菌与早期吞噬体标记物[例如,脑5中的Ras相关蛋白(Rab5)和转铁蛋白受体(TfR) )],以及未获得晚期吞噬体和溶酶体标记物(例如Rab7和LAMP1)(Deretic和Fratti,1999,Mehra等人,2013)。在这里,概述了用分枝杆菌物种感染巨噬细胞的方案,所述分枝杆菌物种如致病性Mtb疫苗菌株牛分枝杆菌 - 卡介苗(BCG)和快速分裂的非致病性耻垢分枝杆菌(Msmeg),然后间接免疫荧光显微镜观察宿主液泡标记。此后,通过使用数学工具处理细菌的二值图像来进行分枝杆菌和宿主液泡标记如TfR和LAMP1之间的共定位程度的自动定量。这导致直接在细菌/细菌簇周围的这些宿主标志物的平均荧光强度(MFI)的定量,相对于手动完成时具有增加的灵敏度。通过操纵宿主或病原体,该测定可用于评价细胞内运输的宿主或细菌决定簇。基本方法可以应用于研究其他细菌或颗粒状珠子的运输,尽管感染和吞噬体成熟的动力学将取决于吞噬性货物。数学分析工具可用于许多标准成像分析程序。然而,对于类似分析的任何适应性应由个体用户利用其成像和分析平台来确认。

关键字:巨噬细胞的感染, 液泡染色, 细菌人口), 吞噬体荧光分析, 形态和布尔逻辑算子

x

材料和试剂

注意:根据机构的实务标准,所有使用现有Mtb的工作都必须在生物安全3级(BSL3)设施中执行

  1. 巨噬细胞,原代巨噬细胞,如C57BL/6骨髓来源的巨噬细胞(BMDM)或巨噬细胞细胞系(如RAW264.7)
    te:BMDM可以如所述分离(Banaiee等人2006; Nagabhushanam等人,2013)。 RAW264.7细胞可以从ATCC(ATCC,目录号:TIB-71)购买。
  2. L929细胞(ATCC,目录号:CCL-1)
  3. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:11965)
  4. 胎牛血清(FBS)(热灭活的)(Life Technologies,Gibco ,目录号:10082147)
  5. 1 M HEPES溶液(Life Technologies,Gibco ,目录号:15630-056)
  6. 200mM L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)
  7. 青霉素 - 链霉素溶液(10,000U/ml)(Life Technologies,Gibco ,目录号:15140-122)
  8. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:10010-023)
  9. 八孔Permanox室玻片(Thermo Fisher Scientific,Nunc Lab-Tek Chamber Slide,目录号:177445)
  10. 八孔腔盖玻片(Thermo Fisher Scientific,Nunc Lab-Tek Chamber coverglass,目录号:155411)
  11. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  12. 牛血清白蛋白(BSA)(级分V)(Thermo Fisher Scientific,目录号:BP1600)
  13. 洗涤剂:皂苷(Sigma-Aldrich,目录号:47036),Triton-X100(Sigma-Aldrich,目录号:X100)和/或Tween-20(Thermo Fisher Scientific,目录号:BP337)
  14. 检测宿主细胞标记的主要抗体
    例如,可以用小鼠抗运铁蛋白受体(抗TfR)抗体(Life Technologies,Invitrogen,目录号:136800)标记回收内体和早期吞噬体。晚期内涵体和溶酶体用兔抗LAMP1抗体(Abcam,目录号:24170)染色。
    注意:
    1. 如果将Mtb感染的载玻片从BSL3中移出用于成像,则所选择的抗体需要在固定之后和如下文在步骤A11中所述的长期固定的灭菌方法下工作,注释a。一些市售抗体可能在长期固定后失去识别或弱识别其表位。 
    2. 在给予弗氏佐剂的动物中不产生多克隆抗体是至关重要的,因为那样它们将直接识别Mtb以及它们所产生的任何细胞标记。应测试所有抗体以验证它们不直接识别Mtb。
  15. 免疫荧光的第二抗体
    第二抗体可针对不同物种和不同颜色获得,并且用户可根据所使用的一抗来选择。它们被多种物质吸附以在免疫染色期间使物种交叉反应性最小化。例如,将山羊抗小鼠Alexa 594(Life Technologies,Molecular Probes,目录号:A11032)和山羊抗兔Alexa 594(Life Technologies,Molecular Probes ,目录号:A11037)。
     注意,Mtb显示自发荧光,当在405nm激发时在475nm处具有发射最大值,因此通过许多DAPI滤光片可视化(Patiño等人,2008)。因此,应选择在该范围内不发荧光的次级抗体。
  16. Lysotracker Red DND-99(DMSO中的1mM储备液)(Life Technologies,Molecular Probes ,目录号:L-7528)
    注意:Lysotracker染料有不同的颜色,可根据颜色要求选择。
  17. 葡聚糖(TexasRed,10,000MW,赖氨酸可固定)(Life Technologies,Molecular Probes ,目录号:D-1863)(在PBS中制备25mg/ml储备液,在-20℃ )
    注意:葡聚糖可以以不同的颜色和分子量获得,并且可以根据需要和实验的目标选择。
  18. Vectashield封固剂(Vector Laboratories,目录号:H-1000)
  19. Middle brook 7H9肉汤(Difco,目录号:271310)
  20. 白蛋白 - 葡萄糖 - 过氧化氢酶(ADC)(BD,目录号:212352)
  21. 油酸 - 白蛋白 - 葡萄糖 - 过氧化氢酶(OADC)(BD,目录号:212351)
  22. 指甲油(透明)
  23. 浸没油(显微镜50CC浸渍油)(例如尼康公司,目录号:IB-MA-MXA20234)
  24. 4%多聚甲醛的PBS溶液(见配方)
  25. DMEM完整媒体(见配方)
  26. DMEM/L929完整媒体(见配方)
  27. L细胞条件培养基(参见配方)
  28. 含有0.1%皂苷的PBS中的2%BSA(参见Recipes)
  29. 含有0.1%triton X-100的PBS(参见Recipes)中的2%BSA
  30. 7H9完整媒体(见配方)
  31. 固定剂(见配方)
  32. 阻塞溶液(参见配方)

设备

  1. 分光光度计(使用比色杯测量分支杆菌培养物在600nm波长下的OD 600光密度)
  2. 一次性1.5ml比色皿(Perfector Scientific,目录号:9003)
  3. 一次性无菌过滤系统(500ml,0.22μm孔径)(Corning,目录号:430758)
  4. 30ml方形培养基瓶(Thermo Fisher Scientific,Nalgene,目录号:NE/2019-0030)
  5. 50ml,15ml falcon管(带塞密封盖)(Corning,目录号:430052和430290)
  6. 盖玻片(22×50mm,厚度#1,0.13-0.17mm)(Thermo Fisher Scientific,目录号:12-545C)
  7. 带有用于旋转细菌培养物的摆动转子的离心机(例如,Beckman Coulter,型号:Allegra X-15R;具有SX4750转子的台式离心机)
    注意:Mtb文化应根据机构实践标准在BSL3设施中处理。
  8. 37℃摇床培养箱,带有用于Mtb液体培养物的气溶胶密封装置
  9. Beckman气雾罐用于在falcon管(例如Beckman Coulter,目录号:BK359232)中离心分枝杆菌培养物。
  10. 多孔板托架盖(例如 Beckman Coulter,更多在此链接上 https://www.beckmancoulter.com/wsrportal/techdocs?docname=GX-BB-012
  11. 37℃摇床培养箱,带有用于Mtb液体培养物的气溶胶密封装置
  12. 荧光显微镜[例如装备有60x的Nikon Eclipse TiE/B型; Plan-Apochromat,NA 1.4油浸物镜,Ti Z驱动,高分辨率单色电荷耦合器件(CCD)数码相机; 光度冷SNAP HQ2和适用于DAPI,FITC和TexasRed频道的滤镜集]

软件

  1. 尼康成像软件 - 元素高级研究(NIS-Elements AR)版本3.2软件与反褶积模块
  2. Graph Pad Prism软件

程序

  1. 巨噬细胞感染
    注意:
    1. 所有使用Live Mtb设置感染(以下步骤A1和A3-11)都应在生物安全级别3(BSL3)工具中完成。
    2. BCG和Msmeg文化应在BSL3设施之外,但在生物安全II级机柜或根据机构实践标准处理。
    3. 应使用合适的气溶胶罐和多孔板盖或根据实践的制度标准,进行Mtb和BCG培养物(下面的步骤A4-6)和感染样品(下面的步骤A9)的离心。这些罐和具有盖的多孔板载体应当在离心期间打开用于在生物安全II级机柜中装载和卸载含有镰刀或感染板的Mtb/BCG培养物。
    1. 从冷冻的细菌原种(从对数中期培养物制备,即在-80℃下在18%甘油中冷冻的OD 600在0.5至1.0单位之间的培养物)接种液体分枝杆菌在10ml的7H9完全培养基中的培养物在30ml方形培养基瓶中。酌情包括抗生素(例如,选择含有卡那霉素作为选择标记的GFP的质粒,包括5μg/ml的卡那霉素)。在37℃下,在90-110rpm下,在气溶胶容纳单元中振荡孵育。 Mtb和BCG在37℃下在7H9完全培养基中大约每20小时双重,所以培养物将需要少量(〜4-5)天达到对数中期。如果需要,可以将培养物稀释到新鲜培养基中(例如,如果使用的抗生素在培养过程中易于降解),则以适当的间隔稀释。 Msmeg大约每3小时翻倍,因此在早晨稀释过夜生长的培养物,以达到对数中期,在白天用于感染。时间将取决于特定的应变,条件和起始接种物
    2. 在组织培养实验室的生物安全II级机柜中感染前一天应进行巨噬细胞的电镀。板细胞在250微升的DMEM或DMEM/L929完全培养基每孔在8井室幻灯片。 BMDM可以每孔1×10 5个细胞的密度接种。如果使用RAW264.6巨噬细胞细胞系,可以在DMEM完全培养基中以6×10 4个/孔的密度接种。将细胞孵育在37 培养箱。 在Mtb感染当天,将幻灯片转移到37 培养箱中,在BSL3设备中具有5%CO 2气氛。
      注意,放置在24孔板中的盖玻片也可以用于对巨噬细胞进行电镀以用于感染。然而,8孔腔室玻片和盖玻片提供无菌和无RNA酶电镀条件,使用最少的试剂。此外,可以用最小孔至孔差异测试多种条件,如在感染之前可以敲低不同的宿主标志物以观察宿主蛋白在吞噬溶酶体运输中的作用或可以在相同载玻片的不同孔中感染多种细菌菌株。
    3. 使用分光光度计在比色杯中测量1ml培养物的OD 600。如果需要稀释培养物,以便在巨噬细胞感染的当天使培养物的OD <600> 在0.5至1.0 OD 600之间。
    4. 将培养物转移到15ml falcon管中,在室温下以1500×g离心5分钟。
    5. 除去上清液并将沉淀重悬于5ml PBS中,并再次在室温下以1500×g离心5分钟。重复一次,以从7H9完全培养基中除去Tween 80
    6. 将沉淀重悬于5ml用于RAW 264.7巨噬细胞的DMEM完全培养基和用于BMDM的DMEM/L929完全培养基中,并将其转移至50ml Falcon。在室温下以450rpm的低速旋转5分钟以通过造粒除去细菌团块。转移上清小心避免沉淀到新鲜的15毫升falcon管。上清液不含具有大量单细胞群体的分枝杆菌群,因此用于感染巨噬细胞。  
      注意:在此步骤中通过沉淀去除〜90%的分枝杆菌是不寻常的。例如,如果在步骤A3中,10ml的Mtb培养物的OD 600为0.5,则预期低速旋转后的上清液具有OD 600至0.05, 0.1,取决于结块的程度。
    7. 测定在步骤A6中收获的上清液的OD 600。将OD <600>转换为细菌数。该值将根据细菌的形状,大小和内部光吸收组分而变化,并且对于相同物种的不同菌株可以是不同的。应该预先确定转换因子。例如,可以如下确定转化因子如OD 600 = 1.0 = 500×10 6个集落形成单位(CFU)/ml。
      1. 一个。将"x"OD 600的起始培养物连续稀释,并将每种稀释液的体积接种以得到CFU。
      2. 给出在可数范围内的菌落的稀释度和该稀释度的菌落数用于使用下式计算"y"CFU/ml:
        CFU/ml =(#菌落)*(稀释因子)/(以ml计的铺板体积)
      3. 这给出x OD 600 = y CFU/ml。现在OD 600为1 = y/x CFU/ml。
    8. 计算以约1-10的感染复数(MOI)感染巨噬细胞所需的步骤A6中制备的细菌悬浮液的体积。将该体积添加到每个孔中。如有必要,进行稀释,使至少10-50μl添加到每个孔中,总体积为200μl。应测试几个MOI,最佳MOI将取决于实验条件的细节。
    9. 在室温下将载玻片在50×g下旋转2分钟,以使用具有在摇摆转子中的盖子的多孔板载体来同步感染。在37中孵育培养箱中,具有5%CO 2气氛。可以在较短的时间段后进行该步骤,虽然细菌摄取将较低
    10. 在所需的时间点[例如感染后3小时(hpi),12hpi和24hpi]除去培养基。 固定在1%PFA/PBS在4℃过夜的Mtb感染的样本或根据研究所的生物安全指南,之前从BSL3删除。 在室温下在4%PFA/PBS中固定15分钟,用于Msmeg和BCG感染的样品。 
      注意:
      1. 根据生物安全规则,Mtb感染的载玻片应该在从BSL3中取出用于免疫染色和成像之前进行灭菌(Schwebach等人,2001)。 这可以通过长固定法实现; 在1%PFA/PBS中固定,在4℃下或根据机构的实践标准过夜(至少12小时)。
      2. 对于替代性固定剂,如甲醇或丙酮,镀敷巨噬细胞和感染的皿应与有机溶剂相容。

  2. 免疫荧光显微镜的染色
    对于免疫荧光(IF)显微镜,必须预优化用于免疫染色的条件,其包括固定剂(参见步骤A11,注释ab),透过细胞的去污剂(triton X-100,皂苷或吐温-20) (Goldenthal等人,1985),对于每个第一抗体进行共定位实验。在标准化过程中,免疫染色的特异性可以通过RNA干扰(RNAi)沉默宿主标志物来证实。重要的是包括将有助于设置获取曝光的对照,并且还在荧光显微镜下用作非特异性染色的对照,如在步骤C21中进一步解释的。对于Mtb感染的样品,应筛选特异性宿主标志物的一级抗体以识别特异性表位,以获得巨噬细胞的最佳灵敏度,该巨噬细胞长期固定以灭菌Mtb,这与短期固定的BCG和Msmeg感染样品相反如协议步骤A11中所述)。
    1. 用300μlPBS洗涤固定液3次。
    2. 封闭:在室温下在封闭缓冲液中孵育1小时。例如,封闭缓冲液可以是含有针对一级抗体优化的0.1%去污剂的PBS中的2%BSA。洗涤剂渗透质膜,使抗体可以进入细胞,BSA阻断以防止非特异性染色。
      对于材料和试剂,转铁蛋白受体(TfR)和LAMP1中提及的市售一抗,分别加入200μl在PBS-0.1%triton X-100中的2%BSA和在PBS-0.1%皂苷中的2%BSA,相应的井被堵。
    3. 一级抗体:向孔中加入150μl稀释的一级抗体,并在4℃下孵育过夜。在标准化的PBS-0.1%洗涤剂中的2%BSA中稀释一抗。对于主要 抗体在材料和方法中提及的,在PBS-0.1%triton X-100和2%BSA的PBS-0.1%皂苷中的2%BSA中稀释TfR(1:250v/v)和LAMP1(1:1,000v/,
    4. 用PBS-0.1%洗涤剂洗涤三次,每次在室温下洗涤5分钟以除去过量的和非特异性结合的抗体,例如使用PBS -0.1%triton X-100转铁蛋白受体和PBS- 0.1%皂苷用于步骤B14中使用的LAMP1抗体
    5. 二抗:根据产生一抗的物种,使用用适当的荧光团标记的适当的抗物种(小鼠或兔)二抗。加入150微升稀释的第二抗体,并在室温下在黑暗中孵育1小时,以防止荧光团漂白。在PBS-0.1%洗涤剂中的2%BSA中稀释第二抗体1:250,例如使用在PBS-0.1%triton X-100中的2%BSA中的抗小鼠Alexa 594来检测抗TfR和稀释的抗兔Alexa 594在含2%BSA的PBS-0.1%皂苷中以检测抗LAMP1。
    6. 重复步骤B15。防止光照,防止二抗的光漂白
    7. 轻轻向上按压,从滑块顶部取出腔室,剥离橡胶垫圈(见图1)
    8. 在载玻片上的8个孔中的每一个的中心放置〜2μl的抗褪色。安装一个干净的矩形盖玻片,并用指甲油密封盖玻片的侧面
    9. 虽然这里没有详细描述,但是可选地,也可以使用lysotracker染料进行溶酶体的活细胞染色或用荧光葡聚糖(例如,TexasRed-葡聚糖)加载它们。活染色的动力学主要有两个变量,浓度和时间,并且根据期望的目标,可以最好地设计实验以将其与感染耦合。提供了对其使用的简要理解:
      1. Lysotracker是一种细胞可渗透的酸性营养染色,可用于标记巨噬细胞中的溶酶体,而不需要固定。染色条件不同,用户可根据实际实验标准化染料浓度和染色时间(30分钟至2小时)。
        1. 平板巨噬细胞和感染他们,如A部分所述。在感染巨噬细胞后所需时间点的最后30分钟,从孔中移除培养基,每孔加入150μl200nM lysotracker,并在37℃孵育。以后保护样品免受光漂白。在处理染色样品时关闭生物安全柜中的灯,以防止光漂白
        2. 用PBS洗涤孔三次,以除去过量的lysotracker。   
        3. 添加PBS,并迅速进行图像采集用荧光显微镜。在无染料培养基中进一步孵育通常导致信号衰退和细胞起泡。
          注意:可以直接采集Msmeg和BCG感染的巨噬细胞样品用于实时图像采集,因此在这种情况下的巨噬细胞应该被镀在8孔室盖玻片上而不是载玻片上,使得室不必被去除。在Mtb感染的巨噬细胞的情况下,在用lysotracker进行活标记后,样品应当在从BSL3中成像之前根据研究所的生物安全指南进行固定。然后可以用PBS洗涤以除去固定剂,并用抗褪色剂装上。
      2. 德克萨斯州葡聚糖(10kD)对细胞的内吞和溶酶体囊泡网络是可自由渗透的,因此可以通过预加载巨噬细胞用于在感染之前标记溶酶体:
        1. 如步骤A2中那样铺板巨噬细胞。从孔中取出电镀培养基,并用150μl的1mg/ml葡聚糖溶液(稀释在PBS中)在37℃处理1小时,通过流体相内吞作用细胞摄取。
        2. 用预温的完全DMEM或DMEM/L929完全培养基(根据需要)洗涤两次,在显微镜下观察,如果需要,用预热的PBS洗涤两次。
        3. 加回温的完全DMEM或DMEM/L929完全培养基(根据需要),并在具有5%CO 2的37℃培养箱中孵育4小时,以将德克萨斯葡聚糖追加到溶酶体中。 这一追逐期可以在隔夜进行,然后第二天感染。
        4. 感染巨噬细胞,并在感染后的所需时间点固定样品
  3. 图像采集
    注意:根据个体或核心设施指南,在操作任何荧光或共聚焦显微镜进行图像采集之前,应先进行适当的训练。
    1. 对于图像采集(有关更多详细信息,请参阅参考文献8),请务必使用以下控件:
      1. 固定但未染色作为细胞和细菌的背景自身荧光的控制
      2. 二次单独控制以检查非特异性染色
      3. 在多色标记实验的情况下的单色控制例如当在相同的孔中用相容的染色程序进行两种不同标记的双重免疫标记时。这对于以与用于获取多色样品的图像完全相同的设置收集所有通道中的每种单一颜色的图像是必要的。它有助于校正串扰(一种染料对于其他染料的入射光的激发)和渗透(一种染料向其他染料的检测器中的发射)。
      4. 如果单色控制指示渗透,最好在每个孔中仅进行一种标记物的免疫标记。
    2. 使用Nikon Eclipse TiE/B显微镜,使用60×1.4NA油浸物镜在高放大率下使用专门用于荧光或共聚焦显微镜的说明来获取图像。设置微分干涉对比度(DIC)和所需颜色通道(DAPI,GFP和/或TxRed)的最佳曝光级别,但不会饱和像素强度。使用具有单色控制的自动曝光工具在估计曝光水平以防止串扰方面非常有用。  
    3. 吞噬后,具有细长杆状形态的分枝杆菌主要定位在细胞的细胞质中的囊泡结构/吞噬体中长达48小时(van der Wel等人,2007)。分枝杆菌的宽度为0.2至0.5μM,平均宽度为0.35μM,因此应当获得合适的z-堆叠。通常应该每孔获得至少三个具有10至15个巨噬细胞的场,以产生每个条件最少100个感兴趣区域(ROI),用于计算统计显着性。 注意:当在DAPI通道中使用Mtb的自发荧光来显现细菌时,建议将样品对于405nm处的激发入射光的暴露保持为最小,因为它快速漂白Mtb的自发荧光。< em>
    4. 如果需要,在使用Nikon Eclipse TiE/B落射荧光显微镜的情况下解卷积图像,并在分析前校正所有图像上的背景。

  4. 图像分析
    内体/溶酶体标记定位在吞噬体货物周围(Fratti等人,2001; Hava等人,2008; Delaby等人, 2012; Mottola 。,2014)。在图2A所示的实施例中,宿主标记物,运铁蛋白受体(TfR)以灰色显示,吞噬体货物Mtb以绿色显示。这里,宿主标记和细菌/细菌簇的共定位的程度在吞噬体之间是可变的(参见图2A中的吞噬体1,2,3和4)。定性分析目视观察吞噬体,2和3具有高和吞噬体,1和4具有围绕细菌的低TfR染色。对于围绕细菌/细菌簇的宿主液泡染色的定量,我描述了使用NIS-Elements AR版本3.2使用图2作为实例进行的自动化分析。
    1. 在细菌的颜色通道中创建二进制图像(绿色)。在二进制图像中,背景将具有0像素强度,并且所选择的对象(细菌)将具有1的像素强度。在给定的示例中,图2B,这可以通过"阈值化"菌。此外,在NIS Elements高级研究软件中,可以使用"自动检测所有"工具,通过检测光标下的像素强度来工作,因此光标移动有助于标记给定字段中的所有细菌。
    2. 使用细菌的二进制图像,"扩张"三次放大活动窗口中的标记细菌。将此图像与扩张的细菌复制到剪贴板,这是一个参考二进制图像,以供以后使用(图2C)。
    3. 现在使用扩张的细菌图像的活动窗口,在步骤D26中,"Erode"三次缩小放大的区域。这将创建一个当前的二进制图像,以供以后使用(图2D)。
    4. 在两个图像(图2E)上使用命令"eXclusive OR"(XOR)函数,分别从步骤D26-27获得参考和当前二进制,以产生处理结果二进制图像,其中在细菌/细菌簇周围标记"环" (图2F)。
    5. 在活动结果二进制图像中,将环转换为感兴趣区域(ROI)(参见图2G中的ROI ID:1-4)。
    6. 打开液泡标记的图像(例如 TfR),并测量该通道中的ROI以获得ROI的平均荧光强度(MFI)(参见图2H)。
      注意:
      1. 分析的验证:图2A中吞噬体2和3上的高共定位和吞噬体1和4上的较低共定位的视觉评分与图2H中的自动化定量相关,具有ROI ID:2,具有最大平均强度和ROI ID :1具有最小的荧光强度。
      2. 对于每个吞噬体货物,对于扩增和侵蚀的计数数目应该由个体用户在一组图像上最佳地确定,以最佳地捕获液泡标记物的信号强度。
    7. 为每个井在Y轴上绘制所有ROI的MFI。图形显示,统计计算和P值可以使用Graph Pad Prism软件确定 注意:
      1. 形状算子(膨胀,腐蚀)和布尔逻辑运算被集成为许多图像分析软件中的处理工具,以便于使用。然而,它们基于复杂的数学理论,因此我参考文献(Serra,1987; Dougherty和Lotufo,2003),要小心这些处理参数的解释。 
      2. 个体用户应该优化对具有吞噬体货物的不同宿主标志物的共定位的自动化分析,并且使用视觉评分对结果进行计数,以如上述方案D节中所述验证分析。
      3. 自动分析贩运实验的结果(例如,在Mtb感染的巨噬细胞中,Mtb与TfR和LAMP1在3和24hpi处的共定位)应当与由盲目观察者手动进行的视觉评分一致。


      图1.显示顶部腔室与8腔室腔室滑动的示意图
      从孔中取出洗涤缓冲液(A),如图所示插入刀片(B),并施加温和向上的压力,这将分离顶部塑料腔(C),露出橡胶垫圈(D)。轻轻剥离橡胶垫圈(E)。



         
      图2.主机标记物与分枝杆菌吞噬体的共定位的自动分析 A.在24hpi下Mtb感染的RAW 264.7巨噬细胞的免疫染色图像。 A:灰色的宿主标记(TfR)和绿色中的Mtb共定位在吞噬体1,2,3,4(由蓝色箭头指示)上至不同程度。图像已被伪彩色的插图。 B-H:如协议部分D的步骤25至30中所述的量化协同定位的二进制图像处理:这里,黑色,灰色和黄色圆圈分别是图像C,D和F的符号表示。 E显示布尔(XOR)算子对图像C和D图像的影响的图示,以产生F.H给出了ID为1至4的不同ROI中TfR的平均荧光强度(MFI)的定量。   

食谱

  1. 4%PBS中的多聚甲醛溶液(pH7.4)(1L)
    多聚甲醛
    40克
    10x PBS 
    100 ml
    去离子水
    800 ml
    注意:多聚甲醛烟雾是有毒的。 所有工作都应在通风柜中进行。
    1. 在热板上将800ml加热至60℃
    2. 在热板上搅拌的同时加入40g多聚甲醛
    3. 加入50μl10N氢氧化钠(NaOH),继续搅拌
    4. 让溶液搅拌直到多聚甲醛溶解。
    5. 加入25ml 10x PBS并混合
    6. 使用适当的pH条测量溶液的pH
    7. 加水至1升的最终体积。
    8. 通过0.45μm过滤器过滤4%多聚甲醛
    9. 等分并在-20°C下冷冻,长期储存。
  2. DMEM完整媒体
    DMEM
    435 ml
    1 M HEPES
    10 ml
    0.2 M L-谷氨酰胺 5 ml
    热灭活胎牛血清50ml
    通过0.2μM过滤器过滤灭菌并在4℃下贮存 如果要用于感染,媒体中不应包括抗生素
  3. DMEM/L929完整媒体
    DMEM                                        
    390 ml
    热灭活FBS
    50 ml
    L929细胞条件培养基
    50 ml
    0.2 M L-谷氨酰胺 5 ml
    0.1M丙酮酸钠 5毫升
    通过0.2μM过滤器过滤灭菌并在4℃下贮存 如果要用于感染,媒体中不应包括抗生素。
  4. 制备L细胞条件培养基
    L929细胞培养基
    DMEM                                                          
    440 ml
    热灭活FBS
    50 ml
    200mM L谷氨酰胺 5 ml
    100x非必需氨基酸
    5 ml
    1,000xβ-巯基乙醇 0.5 ml
    1. 解冻1小瓶的L929细胞,添加1毫升温暖的媒体到小瓶和板在一个15厘米的TC菜至少35毫升温暖的媒体。
    2. 允许成长到汇合,从冷冻股票开始通常需要3-4天
    3. 用1×PBS冲洗细胞,然后用10ml 1x胰蛋白酶EDTA覆盖并在37℃ 1-5分钟,直到细胞以轻轻的移液从板的底部离开。
    4. 向细胞中加入过量的L细胞培养基(3倍体积的胰蛋白酶EDTA加入),并上下吸取以破碎团块。
    5. 以650xg离心5分钟。
    6. 将细胞重悬于25 ml培养基中。
    7. 将1ml细胞加入含有38ml生长培养基的15cm TC培养皿中的25个培养皿中
    8. 允许细胞生长约5-7天,细胞应在第6天达到汇合。吸出培养基并用0.22μM过滤器过滤。
    9. 等分到50毫升的小瓶,并冻结在-80 °C 长达六个月。
  5. 2%BSA的含有0.1%皂苷的PBS(50ml)
    1. 称取0.1g皂苷,溶于100ml 1×PBS中。 过滤0.45μm并在室温下储存
    2. 轻轻地溶解2g BSA在50ml PBS-0.1%皂苷中(新鲜制备)。
  6. 含有0.1%triton X-100(50ml)的2%BSA,
    1. 称取0.1g的triton X-100(w/v),并溶解在100ml的1×PBS中。 过滤0.45μm并在室温下储存
    2. 轻轻地溶解2 g BSA在50 ml PBS-0.1%triton X-100(新鲜制备)。
  7. 7H9完全培养基(1L)
    7H9粉                   
    4.7克
    50%甘油 4.0 ml
    20%吐温80
    2.5 ml 

    到900ml
    1. 将7H9粉末溶解在水中,加入甘油和吐温80. 
    2. 调节水的体积,使最终体积为900ml的介质。
    3. 向Mtb完全培养基中加入100 ml OADC或者为Msmeg和BCG完全培养基加入100 ml ADC。
    4. 通过0.22μM过滤器灭菌并在4°C下储存。  
    5. 或者,在将7H9和甘油溶解在约900ml水中后,高压釜,然后补充吐温-80和100ml ADC/OADC的无菌溶液,并储存在4℃。
  8. 固定剂
    4%PFA/PBS用于固定BCG和Msmeg<
    1%PFA/PBS用于固定Mtb
  9. 封锁解决方案
    含有0.1%洗涤剂的PBS中的2%BSA

致谢

我感谢Jennifer A. Philips对本协议的监督和开发。 该协议改编自公开的工作Mehra等人(2013)。 这项工作得到了NIH(R01 AI087682),Doris Duke慈善基金会,美国传染病学会,Michael Saperstein医学学者研究基金(纽约大学医学院),Potts纪念基金会和 美国微生物学会。

参考文献

  1. Banaiee,N.,Jacobs,W.R.,Jr.and Ernst,J.D。(2006)。 在小鼠肺和巨噬细胞中调节结核分枝杆菌 whiB3。 a> Infect Immun 74(11):6449-6457。
  2. Delaby,C.,Rondeau,C.,Pouzet,C.,Willemetz,A.,Pilard,N.,Desjardins,M.and Canonne-Hergaux,F。 铁和血红素代谢相关蛋白在红细胞生成早期阶段的亚细胞定位。 PLoS One 7(7):e42199。
  3. Deretic,V。和Fratti,R.A。(1999)。 结核分枝杆菌吞噬体 Mol Microbiol 31(6):1603-1609。
  4. Dougherty,E.R。和Lotufo,R.A。(2003)。 实践形态学图像处理。 Spie Bellingham,WA。
  5. Fratti,R.A.,Backer,J.M.,Gruenberg,J.,Corvera,S.and Deretic,V。(2001)。 磷脂酰肌醇3-激酶和Rab5效应子在吞噬体中的作用 生物发生和分枝杆菌吞噬体成熟停滞。细胞生物学154(3):631-644。
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Copyright: © 2014 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. Mehra, A. (2014). Phagolysosomal Trafficking Assay . Bio-protocol 4(13): e1163. DOI: 10.21769/BioProtoc.1163.
  2. Mehra, A., Zahra, A., Thompson, V., Sirisaengtaksin, N., Wells, A., Porto, M., Koster, S., Penberthy, K., Kubota, Y., Dricot, A., Rogan, D., Vidal, M., Hill, D. E., Bean, A. J. and Philips, J. A. (2013). Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog 9(10): e1003734.
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