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Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry
采用流式细胞器细胞术分析噬菌体抗原降解   

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Abstract

Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal degradation of OVA and acquisition of lysosomal proteins (like LAMP-1), can be measured simultaneously in a highly quantitative manner by flow organellocytometry. These read-outs can be correlated to other phagosomal functions, for example antigen degradation, processing and loading in DC.

Background

In innate immunity, professional phagocytes such as dendritic cells (DC), macrophages and neutrophils recognize and internalize different types of particles by phagocytosis including pathogens and dead cells (Flannagan et al., 2012). Intra-phagosomal degradation of these particles by fission and fusion events with endosomal and lysosomal compartments allow either clearance and complete destruction of phagosomal cargo or partial degradation and processing of phagosomal antigens for presentation to T lymphocytes. Different parameters of phagosome maturation, such as acidification, oxidation and proteolysis, dictate phagosomal fate and influence the initiation of different immune responses (Kinchen and Ravichandran, 2008). In particular, the type of involved phagocytes, specific recognition of pathogen-associated molecular patterns (such as LPS) or danger-associated molecular patterns (such as HMGB1) on the surface of the particle as well as the influence of cellular and phagosomal signal transduction determine strength and duration of phagosomal antigen degradation (Savina and Amigorena, 2007).
   This protocol was developed to follow antigen degradation kinetics on the single phagosome level in a highly quantitative fashion by flow organellocytometry. It is based on a method previously published by our lab using antigen-coupled polystyrene beads as phagocytosis model system (Savina et al., 2010). Due to their physical properties, bead-containing phagosomes can be distinguished from other cell organelles of similar size during flow cytometry. Therefore, phagosomal antigen degradation can be measured directly without previous organelle fractionation and purification methods. Another major advantage of this protocol over many other protocols is the fact that it allows to distinguish between internalized beads within phagosomes and particles bound on the outside of the cell, which were not phagocytosed. This protocol was used previously for the characterization of phagosomal antigen degradation in bone marrow-derived DC (BMDC) (Hoffmann et al., 2012; Alloatti et al., 2015) as well as in splenic DC (Alloatti et al., 2015). However, other phagocyte types and different antigen sources can be used as well for the investigation of antigen degradation kinetics in phagosomes. The approach described below is adapted to BMDC and ovalbumin (OVA)-coupled particles as phagosomal cargo.

Materials and Reagents

Note: The entire method is performed with sterile pyrogen-free dishes and plates, pipettes, tips, microfuge and conical tubes. All media and buffers need to be filtered through 0.22 μm filters.

  1. Petri dish, 145 x 20 mm (Greiner Bio One, catalog number: 639161 )
  2. 2 ml tubes
  3. 15 ml centrifuge tube
  4. U-bottom 96-well storage plate (Corning, Falcon®, catalog number: 353077 )
  5. V-bottom 96-well storage plate (Corning, Falcon®, catalog number: 353263 )
  6. 2 ml sterile syringe (Henke Sass Wolf, catalog number: 4020.000V0 )
  7. 22 G sterile needle, 0.7 x 40 mm (Terumo Europe, catalog number: NN-2238R )
  8. Mice: C57BL/6J (Janvier Labs)
  9. Particles: amine-modified polystyrene microspheres, 3 μm diameter (Polysciences, catalog number: 17145-5 )
  10. Dulbecco’s phosphate-buffered saline, no calcium, no magnesium (DPBS) (Thermo Fisher Scientific, GibicoTM, catalog number: 14190094 )
  11. Glutaraldehyde, 25% (vol/vol), EM grade (Electron Microscopy Sciences, catalog number: 16220 )
  12. Low endotoxin ovalbumin (OVA) (Worthington Biochemical, catalog number: LS003062 )
  13. Glycine, 0.5 M in PBS (Biosolve, catalog number: 07132391 )
  14. CO2-independent medium (Thermo Fisher Scientific, GibcoTM, catalog number: 18045088 )
  15. Glutamax supplement (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  16. Iscove’s modified Dulbecco’s medium (IMDM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31980030 )
  17. Low endotoxin fetal bovine serum (FBS, heat-inactivated for 20 min at 56 °C) (Biowest, catalog number: S1860 )
  18. β-mercaptoethanol (50 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
  19. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A4503 )
  21. Anti-chicken egg albumin (OVA antibody) (Sigma-Aldrich, catalog number: C6534 )
  22. Purified rat anti-mouse CD16/CD32 monoclonal antibody (Fc block) (BD, PharmingenTM, catalog number: 553142 )
  23. Anti-mouse LAMP-1 antibody, biotin conjugate (Affymetrix, eBioscience, catalog number: 13-1071-82 )
  24. Goat anti-rabbit IgG (H+L) antibody, DyLight 633 conjugate (Thermo Fisher Scientific, InvitrogenTM, catalog number: 35562 )
  25. Goat anti-rabbit IgG (H+L) antibody, Alexa Fluor 568 conjugate (Thermo Fisher Scientific, InvitrogenTM, catalog number: A11036 )
  26. Streptavidin, Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S11223 )
  27. Imidazole (Sigma-Aldrich, catalog number: I-0250 )
  28. Sucrose (EMD Millipore, catalog number: 107651 )
  29. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P-7626 )
  30. EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog number: 11873580001 )
  31. Dithiothreitol (DTT) (EMD Millipore, catalog number: 233155 )
  32. Trypan blue solution, 0.4% (wt/vol) (MP Biomedicals, catalog number: 0916910 )
  33. Internalization medium (see Recipes)
  34. BMDC culture medium (see Recipes)
  35. Homogenization buffer (see Recipes)

Equipment

  1. Vortex
  2. Refrigerated centrifuge for tubes of 2 ml, 15 ml and 50 ml size as well as 96-well plates
  3. Tissue culture incubator adjusted to 37 °C and 5% CO2
  4. Stuart test tube rotator wheel tolerating 4 °C (Bibby Scientific, model: SB3 )
  5. Temperature-controlled water bath
  6. Pipets
  7. Tissue culture light microscope equipped with bright field and 20x objective
  8. Multi-channel pipet
  9. LSR II flow cytometer (BD) or any other multicolour flow cytometer

Software

  1. FlowJo software (FlowJo, LLC.)
  2. Prism 6 software (GraphPad Software, Inc.)

Procedure

  1. Preparation of antigen-coupled particles
    Note: Estimate the number of cells, which will be used for this assay, in advance to prepare a sufficient amount of particles coupled to OVA the day before the experiment. The stock suspension of 3 μm microspheres contains 1.68 x 109 particles/ml.
    Example: Estimate the amount of cells you will harvest based on the amount of dishes in culture (for example 20 x 106 cells in total from four 145 mm dishes of BMDC). Multiply this number by 10 (the amount of beads provided per cell) and divide by the concentration of beads (1.68 x 109 particles/ml).

                     
    1. Vortex suspension of amine-modified polystyrene microspheres thoroughly and distribute into 2 ml tubes (max. 200 μl/tube).
    2. Add 1.6 ml PBS to each tube, vortex and spin for 3 min at 16,000 x g at 4 °C.
    3. Discard supernatant, resuspend the pellet in 1.8 ml PBS and vortex. Spin again.
    4. Discard supernatant and resuspend the pellet in 8% glutaraldehyde/PBS (vol/vol). Use five times the volume of beads, which was used initially (e.g., 200 μl in 1 ml).
    5. Vortex and combine all suspensions in a 15 ml centrifuge tube. Protect the tube from light and incubate for 4 h on a test-tube rotator at 20 rpm at room temperature.
    6. Transfer suspension back into 2 ml tubes and spin them for 3 min at 16,000 x g at 4 °C.
    7. Discard supernatant, resuspend and vortex pellet in 1.8 ml PBS. Spin again.
    8. Discard supernatant and resuspend the pellet in 0.5 mg/ml OVA/PBS (Use five times the volume of beads that was used initially [e.g., 200 μl in 1 ml]).
    9. Vortex and combine all suspensions in a 15 ml centrifuge tube. Incubate overnight on a test-tube rotator at 20 rpm at 4 °C.
    10. Transfer suspension back into 2 ml tubes and spin them for 3 min at 16,000 x g at 4 °C.
    11. Discard supernatant, resuspend and vortex pellet in 1.8 ml 0.5 M glycine/PBS.
    12. Incubate for 30 min on a test-tube rotator at 20 rpm at 4 °C.
    13. Spin tubes for 3 min at 16,000 x g at 4 °C.
    14. Discard supernatant, resuspend and vortex pellet in 1.8 ml PBS.
    15. Repeat the spin followed by two more washes with 1.8 ml PBS (a total of three washes).
    16. Resuspend each pellet in the initial volume (200 μl), pool and vortex the dispersion. Store the suspension of OVA-coupled microspheres at 4 °C.
      Note: Coupled microspheres should be prepared freshly the day before the flow organellocytometry experiment and can be used within a week when stored at 4 °C. Never freeze particles and always vortex dispersion carefully to avoid aggregation of coupled microspheres.

  2. Phagocytosis of antigen-coupled particles
    Note: Estimate around 5 x 106 cells for each experimental condition.
    1. Pick up cell clusters and single cells by resuspending them with a pipet. Spin cells for 4 min at 400 x g at 4 °C.
    2. Resuspend cells in ice-cold PBS and spin them again for 4 min at 400 x g at 4 °C.
    3. Resuspend cells in ice-cold internalization medium, transfer them to a conical 15 ml centrifuge tube and count the cells.
    4. Adjust the cell suspension in ice-cold internalization medium to a cell density of 20 x 106/ml and keep them on ice.
    5. Vortex suspension of OVA-coupled microspheres and add them to the cells at a particle-to-cell ratio of 10:1. Mix carefully.
    6. Incubate samples for 30 min in a water bath adjusted to 16 °C. If necessary, add ice to the water bath during the incubation period.
    7. Place samples on ice and add 10 ml ice-cold PBS to the tubes. Pipet up and down, close the tubes and shake them thoroughly for 10 sec. Spin tubes for 4 min at 100 x g at 4 °C to remove floating particles.
    8. Discard supernatant and repeat the last step twice to have a total of three washes.
    9. Resuspend each cell pellet in pre-warmed BMDC culture medium and divide cell suspension in 15 ml centrifuge tubes to allow phagosomal antigen degradation to occur for different time points. Use 1-2 ml medium for each experimental condition.
    10. Add 10 ml ice-cold PBS to one tube, which represents the time point of 0 min. Keep this tube on ice.
    11. Keep the other tubes open and incubate them at 37 °C and 5% CO2 for different chase periods (for example: 60 and 120 min).
    12. Stop each chase period by adding 10 ml of ice-cold PBS and keep these tubes on ice.

  3. Manual lysis and labeling of samples
    Note: After phagocytic uptake of particles, cells undergo a labeling step that distinguishes surface-bound particles from internalized ones. Subsequently, cells are lysed manually to release cytosol and cell organelles followed by a labeling step to detect levels of phagosomal OVA as well as the acquisition of lysosomal markers (for example, LAMP-1) to phagosomes during phagosome maturation.
    1. Spin all tubes for 4 min at 400 x g at 4 °C.
    2. Discard supernatant and resuspend each pellet in PBS + 1% (wt/vol) BSA. Transfer samples to a U-bottom 96-well plate.
    3. Spin the plate for 4 min at 400 x g at 4 °C and flick the supernatant.
    4. Resuspend each sample in 0.2 ml PBS + 1% (wt/vol) BSA + 1:100 diluted CD16/CD32 antibody (Fc block). Incubate on ice for 10 min.
    5. Spin the plate for 4 min at 400 x g at 4 °C and flick the supernatant.
    6. Resuspend each sample in 0.2 ml PBS + 1% (wt/vol) BSA + 1:500 diluted OVA antibody. Incubate on ice for 15 min.
    7. Spin the plate for 4 min at 400 x g at 4 °C and flick the supernatant.
    8. Resuspend each sample in 0.2 ml PBS and spin the plate for 4 min at 400 x g at 4 °C. Flick the supernatant and repeat the wash.
    9. Resuspend each sample in 0.2 ml PBS + 1% (wt/vol) BSA + 1:1,000 diluted anti-rabbit Alexa Fluor 568. Incubate on ice for 15 min.
    10. Spin the plate for 4 min at 400 x g at 4 °C and flick the supernatant.
    11. Resuspend each sample in 0.2 ml PBS and spin the plate for 4 min at 400 x g at 4 °C. Flick the supernatant and repeat the wash.
    12. Transfer samples into conical 1.5 ml centrifuge tubes and spin them for 4 min at 400 x g at 4 °C.
    13. Aspirate the supernatant carefully and resuspend each cell pellet in 0.5 ml homogenization buffer.
    14. For mechanical lysis of the cells, pass the suspension 15 times thoroughly through a 22 G needle fitted to a 2 ml syringe. Use the whole volume of the syringe for mechanical breaking of cells. Control homogenization success by trypan blue staining (Figure 1): Add 1 μl sample to 9 μl 0.4% (wt/vol) trypan blue solution, mix and add to a cell culture counting chamber. Count the amount of unstained, intact cells as well as stained, lysed cells using a light microscope. Calculate the percentage of lysed cells and do not exceed 80% of lysed cells. Additionally, avoid breaking of cell nuclei.


      Figure 1. Mechanical lysis of BMDC after uptake of OVA-coupled beads to release phagosomes for subsequent flow organellocytometry. BMDC were allowed to internalize OVA-coupled beads for 30 min followed by a chase period to allow phagosome maturation to occur. To break cells mechanically in order to release intact phagosomes without breaking cell nuclei, we pass the cell suspension 15 times through a 22 G needle. The image shows the cell suspension labeled with trypan blue before (upper panel) and after cell lysis (lower panel). Accumulation of trypan blue in cells with broken cell membrane but intact nuclei (arrows) allows one to monitor success of mechanical cell lysis. The release of phagosomes in the obtained post-nuclear supernatant (PNS) is visualized by bright field microscopy. Scar bar = 20 μm.

    15. Spin the tubes for 4 min at 150 x g at 4 °C to separate the post-nuclear supernatant (PNS) from cell nuclei and remaining intact cells.
    16. Transfer the PNS of the different samples into a V-bottom 96-well plate and keep on ice.
    17. Spin the plate for 3 min at 1,500 x g at 4 °C and remove supernatant with a multi-channel pipet.
    18. Add 50 μl PBS + 1% (wt/vol) BSA + 1:500 diluted OVA antibody + 1:100 diluted LAMP-1 antibody.
    19. Mix samples with a multi-channel pipet, seal the plate and incubate overnight on ice.
    20. Add 0.15 ml PBS + 0.1% (wt/vol) BSA to each well, spin the plate for 3 min at 1,500 x g at 4 °C and remove supernatant with a multi-channel pipet.
    21. Add 0.2 ml PBS + 0.1% (wt/vol) BSA and mix samples with a multi-channel pipet.
    22. Spin the plate again and repeat the wash with PBS + 0.1% (wt/vol) BSA once more.
    23. After the supernatant is removed with a multi-channel pipet, add 50 μl PBS + 1% (wt/vol) BSA + 1:1,000 diluted anti-rabbit DyLight 633 + 1:1,000 diluted streptavidin Alexa Fluor 488. Incubate on ice for 45 min.
    24. Add 0.15 ml PBS + 0.1% (wt/vol) BSA to each well, spin the plate for 3 min at 1,500 x g at 4 °C and remove supernatant with a multi-channel pipet.
    25. Add 0.2 ml PBS + 0.1% (wt/vol) BSA and mix samples with a multi-channel pipet.
    26. Spin the plate again and repeat the wash with PBS + 0.1% (wt/vol) BSA once more.
      Resuspend each sample in 0.2 ml PBS and keep the plate on ice until the measurement by flow cytometry. Measure the samples in a non-fixed state on the same day.  

Data analysis

Note: After the PNS, which contains the phagosomes, has been labeled for OVA and LAMP-1, it is analyzed by flow organellocytometry to determine the kinetics of antigen degradation (level of phagosomal OVA at a given time point) and of the acquisition of lysosomal markers (e.g., LAMP-1). Due to the physical properties and the specific size of microsphere-containing phagosomes, a gating strategy can be applied to measure these parameters simultaneously.

  1. Measure the samples by multicolour flow cytometry and apply the following gating strategy:
    1. Determine the population of beads and phagosomes in the PNS by measuring OVA-coupled beads alone and apply similar forward scatter (FSC) and side scatter (SSC) settings to your samples. Set a gate on the single particle population (Figure 2A).
    2. Exclude surface-bound beads (that are labeled before manual lysis) from subsequent analysis. Include only phagosomes, which are negative for OVA detected by anti-rabbit Alexa 568, by applying a second gate (Figure 2B).
    3. The gated population contains single phagosomes, which can be analyzed over time for OVA degradation and LAMP-1 acquisition either simultaneously (Figure 2C) or separately (Figure 2D) by plotting histograms of OVA (detected by anti-rabbit DyLight 633) and LAMP-1 (detected by streptavidin Alexa Fluor 488).
      Note: Depending on the applied fluorophores for antibody labeling and the used flow cytometer, compensation between the different channels might be necessary.
  2. Analysis of flow cytometry data was performed using FlowJo software (FlowJo, LLC.). Statistical analysis was performed using Prism 6 software (GraphPad Software, Inc.). Each single experiment needs to be repeated by a sufficient number of independent experiments (at least in triplicate) to draw conclusions from the flow organellocytometry analysis. The data shown in Figure 2 is representative of at least three independent experiments.


    Figure 2. Gating strategy of the applied flow organellocytometry analysis of DC phagosomes. LPS-treated BMDC were allowed to internalize OVA-coated beads at 16 °C and incubated for different chase periods (0 min, 60 min, 120 min) at 37 °C to allow phagosome maturation. Surface-bound, non-internalized beads were stained with anti-OVA and an Alexa 568-coupled secondary antibody. Subsequently, cells were mechanically lysed to release the post-nuclear supernatant (PNS) including bead-containing phagosomes. The PNS was stained for OVA (detected by a DyLight 633-coupled secondary antibody) and LAMP-1 (detected by an Alexa 488-coupled secondary antibody) and analyzed simultaneously by flow cytometry. A. The first gate was set on single beads and single bead-containing phagosomes using forward scatter (FSC) and side scatter (SSC) settings of the labeled PNS. B. In a second gate, non-internalized beads (positive for Alexa 568) were separated from bead-containing phagosomes (negative for Alexa 568). C. Phagosomes were analyzed simultaneously for degradation of OVA and acquisition of LAMP-1 by applying a third gate on the mature, OVA (Dylight 633)-negative and LAMP-1 (Alexa 488)-positive population. D. Alternatively, these two parameters of phagosome maturation can also be analyzed separately and quantified over time.

Notes

  1. BMDC are generated from isolated murine bone marrow progenitor cells in GM-CSF-containing medium for 9 days. On the day of experiment, cell surface expression of CD11c should always exceed 85%.
  2. The reproducibility of results is highly dependent on homogenous conditions during mechanical breaking and cell lysis. Always control the success of cell lysis by trypan blue staining. Another factor is the phagocytic efficiency of the used cells. Always count cell numbers of your samples to apply comparable bead-to-cell ratios during bead uptake. Do not exceed this ratio, because high numbers of internalized particles will induce cell death and influence the parameters that are measured by flow organellocytometry.
  3. Although OVA-coupled particles are used here as phagocytosis model system, other ligands can be applied during bead coupling to investigate the influence of receptor-ligand interactions on phagosome maturation. Some examples are published elsewhere (Hoffmann et al., 2010; Hoffmann et al., 2012).

Recipes

  1. Internalization medium
    CO2-independent medium
    1x glutamax supplement
  2. BMDC culture medium
    IMDM
    10% (vol/vol) FBS
    50 μM β-mercaptoethanol
    100 IU/ml penicillin
    100 μg/ml streptomycin
    10% (vol/vol) supernatant from J558 plasmacytoma cells, which was used as GM-CSF source (Winzler et al., 1997)
  3. Homogenization buffer
    3 mM imidazole, pH 7.4
    250 mM sucrose
    2 mM PMSF
    1x protease inhibitor cocktail
    2 mM DTT

Acknowledgments

This work was supported by the French National Research Agency through the ‘Investments for the Future’ program (France-BioImaging, ANR-10-INSB-04), ANR-11-LABX-0043 and by the CelTisPhyBio Labex (N- ANR-10-LBX-0038), part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL). We are grateful to the financial support by the European Research Council (2013-AdG No.340046 DCBIOX), by La Ligue Nationale contre le Cancer (EL2014.LNCC/SA), by Fonds Wetenschappelijk Onderzoek (FWO; 1526615N; 11W8415N), by an EMBO long-term fellowship (ALTF 883-2011) and by fellowships of Fondation Recherche Médicale (SPF20101221176) and the omics@VIB program (co-financed by the Marie Curie FP7 People Cofund).
The protocol described here is based on a previously published protocol by our lab (Savina et al., 2010), which was developed further to measure different phagosome maturation parameters in dendritic cells and macrophages.

References

  1. Alloatti, A., Kotsias, F., Pauwels, A. M., Carpier, J. M., Jouve, M., Timmerman, E., Pace, L., Vargas, P., Maurin, M., Gehrmann, U., Joannas, L., Vivar, O. I., Lennon-Dumenil, A. M., Savina, A., Gevaert, K., Beyaert, R., Hoffmann, E. and Amigorena, S. (2015). Toll-like receptor 4 engagement on dendritic cells restrains phago-lysosome fusion and promotes cross-presentation of antigens. Immunity 43(6): 1087-1100.
  2. Flannagan, R. S., Jaumouille, V. and Grinstein, S. (2012). The cell biology of phagocytosis. Annu Rev Pathol 7: 61-98.
  3. Hoffmann, E., Kotsias, F., Visentin, G., Bruhns, P., Savina, A. and Amigorena, S. (2012). Autonomous phagosomal degradation and antigen presentation in dendritic cells. Proc Natl Acad Sci U S A 109(36): 14556-14561.
  4. Hoffmann, E., Marion, S., Mishra, B. B., John, M., Kratzke, R., Ahmad, S. F., Holzer, D., Anand, P. K., Weiss, D. G., Griffiths, G. and Kuznetsov, S. A. (2010). Initial receptor-ligand interactions modulate gene expression and phagosomal properties during both early and late stages of phagocytosis. Eur J Cell Biol 89(9): 693-704.
  5. Kinchen, J. M. and Ravichandran, K. S. (2008). Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol 9(10): 781-95.
  6. Savina, A. and Amigorena, S. (2007). Phagocytosis and antigen presentation in dendritic cells. Immunol Rev 219: 143-156.
  7. Savina, A., Vargas, P., Guermonprez, P., Lennon, A. M. and Amigorena, S. (2010). Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays. Methods Mol Biol 595: 383-402.
  8. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L., Zimmermann, V. S., Davoust, J. and Ricciardi-Castagnoli, P. (1997). Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med 185(2): 317-328.

简介

专业吞噬细胞通过吞噬作用内化自身和非自身颗粒以启动先天免疫应答。内化后,形成的吞噬体通过与内体和溶酶体的融合和裂变事件成熟,以获得更酸性,氧化和水解的环境用于其货物的降解。有趣的是,吞噬体成熟动力学在细胞类型和细胞活化状态之间不同。该协议允许使用流式细胞术定量不同类型的吞噬细胞中单个细胞器水平上的吞噬体成熟动力学。在这里,卵白蛋白(OVA)耦合的颗粒用作吞噬作用模型系统在树突状细胞(DC),其通过吞噬内化。在不同的时间点之后,吞噬体成熟参数,例如OVA的吞噬体降解和溶酶体蛋白(例如LAMP-1)的获得,可以通过流式细胞器细胞计数以高度定量的方式同时测量。这些读出可以与其他吞噬体功能相关,例如抗原降解,在DC中的加工和负载。

[背景] 在先天免疫中,专业吞噬细胞如树突细胞(DC),巨噬细胞和嗜中性粒细胞通过吞噬作用识别和内化不同类型的颗粒,包括病原体和死细胞Flannagan等人,2012)。这些颗粒通过与内体和溶酶体区室的裂变和融合事件的吞噬体内降解允许清除和完全破坏吞噬体货物或部分降解和加工吞噬体抗原以呈递给T淋巴细胞。吞噬体成熟的不同参数,如酸化,氧化和蛋白水解,决定吞噬体的命运和影响不同免疫反应的启动(Kinchen和Ravichandran,2008)。特别地,涉及的吞噬细胞的类型,在病原体相关分子模式(例如LPS)或危险相关分子模式(例如HMGB1)在颗粒的表面上的特异性识别以及细胞和吞噬体信号转导的影响确定噬菌体抗原降解的强度和持续时间(Savina和Amigorena,2007)。
   该方案被开发以通过流式细胞器细胞计数以高度定量的方式遵循单吞噬体水平上的抗原降解动力学。它基于我们实验室以前使用抗原偶联的聚苯乙烯珠作为吞噬模型系统发布的方法(Savina等人,2010)。由于它们的物理性质,在流式细胞术期间,含珠吞噬体可以与相似大小的其他细胞器官区分开。因此,吞噬体抗原降解可以直接测量而不需要预先的细胞器分馏和纯化方法。该协议相对于许多其它协议的另一个主要优点是其允许区分吞噬体内的内化珠和细胞外部结合的颗粒,所述颗粒未被吞噬。该方案以前用于表征骨髓衍生的DC(BMDC)中的吞噬体抗原降解(Hoffmann等人,2012; Alloatti等人,2015)以及在脾DC中(Alloatti等人,2015)。然而,其他吞噬细胞类型和不同的抗原来源也可以用于研究吞噬体中的抗原降解动力学。下面描述的方法适用于作为吞噬体货物的BMDC和卵清蛋白(OVA)偶联的颗粒。

材料和试剂

注意:整个方法使用无菌无热原的培养皿和板,移液管,吸头,微量离心管和锥形管进行。所有介质和缓冲液需要通过0.22μm过滤器过滤。

  1. 培养皿,145×20mm(Greiner Bio One,目录号:639161)
  2. 2 ml管
  3. 15ml离心管
  4. U底96孔培养板(Corning,Falcon ,目录号:353077)
  5. V底96孔培养板(Corning,Falcon ,目录号:353263)
  6. 2ml无菌注射器(Henke Sass Wolf,目录号:4020.000V0)
  7. 22G无菌针,0.7×40mm(Terumo Europe,目录号:NN-2238R)
  8. 小鼠:C57BL/6J(Janvier Labs)
  9. 颗粒:直径3μm的胺改性聚苯乙烯微球(Polysciences,目录号:17145-5)
  10. Dulbecco's磷酸盐缓冲盐水,无钙,无镁(DPBS)(Thermo Fisher Scientific,Gibico TM,目录号:14190094)
  11. 戊二醛,25%(vol/vol),EM级(Electron Microscopy Sciences,目录号:16220)
  12. 低内毒素卵清蛋白(OVA)(Worthington Biochemical,目录号:LS003062)
  13. 甘氨酸,0.5M的PBS(Biosolve,目录号:07132391)
  14. CO 2独立培养基(Thermo Fisher Scientific,Gibco TM ,目录号:18045088)。
  15. Glutamax补充物(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  16. Iscove改良的Dulbecco培养基(IMDM)(Thermo Fisher Scientific,Gibco TM ,目录号:31980030)
  17. 低内毒素胎牛血清(FBS,在56℃热灭活20分钟)(Biowest,目录号:S1860)
  18. β-巯基乙醇(50mM)(Thermo Fisher Scientific,Gibco TM ,目录号:31350010)
  19. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  20. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503)
  21. 抗鸡卵清蛋白(OVA抗体)(Sigma-Aldrich,目录号:C6534)
  22. 纯化的大鼠抗小鼠CD16/CD32单克隆抗体(Fc区)(BD,Pharmingen TM ,目录号:553142)
  23. 抗小鼠LAMP-1抗体,生物素偶联物(Affymetrix,eBioscience,目录号:13-1071-82)
  24. 山羊抗兔IgG(H + L)抗体,DyLight 633缀合物(Thermo Fisher Scientific,Invitrogen TM,目录号:35562)
  25. 山羊抗兔IgG(H + L)抗体,Alexa Fluor 568缀合物(Thermo Fisher Scientific,Invitrogen TM,目录号:A11036)
  26. 链霉亲和素,Alexa Fluor?488缀合物(Thermo Fisher Scientific,Molecular Probes TM,目录号:S11223)
  27. 咪唑(Sigma-Aldrich,目录号:I-0250)
  28. 蔗糖(EMD Millipore,目录号:107651)
  29. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P-7626)
  30. 无EDTA蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:11873580001)
  31. 二硫苏糖醇(DTT)(EMD Millipore,目录号:233155)
  32. 台盼蓝溶液,0.4%(wt/vol)(MP Biomedicals,目录号:0916910)
  33. 内部化媒介(见配方)
  34. BMDC培养基(见配方)
  35. 均质缓冲液(参见配方)

设备

  1. 涡流
  2. 用于2ml,15ml和50ml大小的管以及96孔板的冷冻离心机
  3. 将调节至37℃和5%CO 2 的组织培养孵育器
  4. Stuart试管旋转轮耐受4°C(Bibby Scientific,型号:SB3)
  5. 温控水浴
  6. 邮递
  7. 组织培养光学显微镜配备明场和20倍物镜
  8. 多通道移液器
  9. LSR II流式细胞仪(BD)或任何其他多色流式细胞仪

软件

  1. FlowJo软件(FlowJo,LLC。)
  2. Prism 6软件(GraphPad Software,Inc。)

程序

  1. 抗原偶联颗粒的制备
    注意:估计将用于该测定的细胞数,以预先在实验前一天制备足够量的与OVA偶联的颗粒。 3μm微球的原料悬浮液含有1.68×10 9颗粒/ml。
    实施例:基于培养物中的培养皿的量估计要收获的细胞的量(例如,从四个145mm的BMDC培养皿中总共20×10 6个细胞)。将该数乘以10(每个细胞提供的珠的量),并除以珠的浓度(1.68×10 9个/ml颗粒)。

                      
    1. 彻底将胺改性的聚苯乙烯微球的涡旋悬浮液并分配到2ml管(最大200μl/管)中
    2. 向每个管中加入1.6ml PBS,在4℃下以16,000×g旋转并旋转3分钟。
    3. 弃去上清液,将沉淀重悬于1.8ml PBS中并涡旋。再次旋转。
    4. 弃去上清液,并在8%戊二醛/PBS(vol/vol)中重悬沉淀。使用五倍体积的珠,其最初使用(例如,在1ml中200μl)。
    5. 涡旋并将所有悬浮液混合在15ml离心管中。保护管不受光,并在试管旋转器上在室温下以20rpm孵育4小时
    6. 将悬浮液转移回2ml管中,并在4℃下以16,000×g旋转3分钟。
    7. 弃去上清液,在1.8ml PBS中重悬和涡流沉淀。再次旋转。
    8. 弃去上清液并在0.5mg/ml OVA/PBS中重悬沉淀物(使用最初使用的5倍体积的珠子[例如,在1ml中200μl])。
    9. 涡旋并将所有悬浮液混合在15ml离心管中。在试管旋转器上在4℃下以20rpm孵育过夜
    10. 将悬浮液转移回2ml管中,并在4℃下以16,000×g旋转3分钟。
    11. 弃去上清液,在1.8ml 0.5M甘氨酸/PBS中重悬和涡旋沉淀
    12. 在试管旋转器上以20rpm在4℃下孵育30分钟
    13. 在4℃下以16,000×g离心3分钟。
    14. 弃去上清液,在1.8ml PBS中重悬和涡流沉淀
    15. 重复旋转,然后用1.8ml PBS再洗涤两次(总共三次洗涤)
    16. 在初始体积(200μl)中重悬每个沉淀,汇集并涡旋分散体。将OVA偶联的微球的悬浮液储存在4℃ 注意:偶联的微球应在流式细胞器测定实验前一天新鲜制备,并且在4℃储存时可在一周内使用。切勿冻结颗粒,并经常仔细涡旋分散,以避免偶联的微球聚集
  2. 抗原偶联颗粒的吞噬作用
    注意:对于每个实验条件,估计大约5 x 10 6个单元格。
    1. 通过用移液管重悬细胞簇和单细胞。在4℃下以400×g离心细胞4分钟。
    2. 在冰冷的PBS中重悬细胞,并在4℃下以400×g离心4分钟。
    3. 在冰冷的内化培养基中重悬细胞,将其转移到锥形15ml离心管中并计数细胞。
    4. 在冰冷的内化培养基中将细胞悬浮液调节至20×10 6个/ml的细胞密度,并将其保持在冰上。
    5. OVA偶联的微球的涡旋悬浮液,并将它们以10:1的颗粒与细胞比率添加至细胞。小心混合。
    6. 在调节至16℃的水浴中孵育样品30分钟。如有必要,在孵化期间向水浴中加入冰
    7. 将样品置于冰上,向管中加入10ml冰冷的PBS。吸移上下,关闭管,并彻底摇动他们10秒。在4℃下以100×g离心4分钟以除去浮动颗粒
    8. 弃去上清液,重复最后一步两次,共进行三次洗涤
    9. 将每个细胞沉淀重悬在预热的BMDC培养基中,并将细胞悬浮液在15ml离心管中,以允许吞噬体的抗原降解发生在不同的时间点。每个实验条件使用1-2毫升培养基。
    10. 向一个管中加入10ml冰冷的PBS,这表示0分钟的时间点。将此管保持在冰上。
    11. 保持其他管开放,并在37℃和5%CO 2下孵育不同的追踪期(例如:60和120分钟)。
    12. 停止每个追逐期,加入10毫升冰冷的PBS,并保持这些管在冰上
  3. 手动裂解和标记样品
    注意:吞噬吞噬颗粒后,细胞经历标记步骤,区分表面结合颗粒与内化的颗粒。随后,手动裂解细胞以释放细胞质和细胞器,然后进行标记步骤以检测吞噬体OVA的水平以及在吞噬体成熟期间获得溶酶体标记物(例如,LAMP-1)至吞噬体。
    1. 在4℃下,在400×g下旋转所有管4分钟。
    2. 弃去上清液,并在PBS + 1%(wt/vol)BSA中重悬每个沉淀。将样品转移到U底96孔板。
    3. 在4℃下以400×g离心该板4分钟,并轻轻上清液。
    4. 将每个样品重悬于0.2ml PBS + 1%(wt/vol)BSA + 1:100稀释的CD16/CD32抗体(Fc块)中。在冰上孵育10分钟。
    5. 在4℃下在400×g下旋转平板4分钟,并轻轻上清。
    6. 将每个样品重悬于0.2ml PBS + 1%(wt/vol)BSA + 1:500稀释的OVA抗体中。在冰上孵育15分钟。
    7. 在4℃下,在400×g下旋转板4分钟,并轻轻上清。
    8. 将每个样品重悬于0.2ml PBS中,并在4℃下以400×g离心该板4分钟。轻轻上清液并重复洗涤。
    9. 将每个样品重悬于0.2ml PBS + 1%(wt/vol)BSA + 1:1,000稀释的抗兔Alexa Fluor 568中。在冰上孵育15分钟。
    10. 在4℃下在400×g下旋转板4分钟,并轻轻上清。
    11. 将每个样品重悬于0.2ml PBS中,并在4℃下以400×g离心该板4分钟。轻轻上清液并重复洗涤。
    12. 将样品转移到锥形1.5ml离心管中,并在4℃下以400×g离心4分钟。
    13. 小心吸出上清液,并将每个细胞沉淀重悬在0.5ml匀浆缓冲液中
    14. 对于细胞的机械裂解,将悬浮液彻底通过装配至2ml注射器的22G针头15次。使用注射器的整个体积机械破碎细胞。通过台盼蓝染色(图1)控制匀浆成功:将1μl样品加入到9μl0.4%(wt/vol)台盼蓝溶液中,混合并加入细胞培养计数室。使用光学显微镜计数未染色的完整细胞以及染色的裂解细胞的量。计算裂解细胞的百分比,不超过裂解细胞的80%。此外,避免破坏细胞核。


      图1.在吸收OVA偶联的珠粒以释放吞噬体用于随后的流式细胞器血细胞计数之后的BMDC的机械裂解。使BMDC内化OVA偶联的珠子30分钟,随后为追踪期以允许吞噬体成熟发生。为了机械破碎细胞以释放完整的吞噬体而不破坏细胞核,我们将细胞悬浮液通过22G针15次。该图显示了在(上图)和细胞裂解后(下图)用台盼蓝标记的细胞悬浮液。在细胞膜破碎但细胞核完整(箭头)的细胞中积累的台盼蓝允许人们监测机械细胞裂解的成功。通过明视野显微镜观察获得的后核上清液(PNS)中吞噬体的释放。标尺=20μm。

    15. 在4℃下在150×g下旋转管4分钟,以将细胞核上清液(PNS)与细胞核分离并保留完整的细胞。
    16. 将不同样品的PNS转移到V型底96孔板中并保存在冰上
    17. 在4℃下以1,500×g离心该板3分钟,并用多通道移液管除去上清液。
    18. 加入50μlPBS + 1%(wt/vol)BSA + 1:500稀释的OVA抗体+ 1:100稀释的LAMP-1抗体。
    19. 用多通道移液器混合样品,密封板并在冰上孵育过夜。
    20. 向每个孔中加入0.15ml PBS + 0.1%(wt/vol)BSA,在4℃下以1,500xg离心该板3分钟,并用多通道移液管除去上清液。 >
    21. 加入0.2ml PBS + 0.1%(wt/vol)BSA,并用多通道移液器混合样品
    22. 再次旋转板,并再次用PBS + 0.1%(wt/vol)BSA重复洗涤。
    23. 用多通道移液管除去上清液后,加入50μlPBS + 1%(wt/vol)BSA + 1:1,000稀释的抗兔DyLight 633 + 1:1,000稀释的链霉亲和素Alexa Fluor 488.在冰上孵育45分钟min。
    24. 向每个孔中加入0.15ml PBS + 0.1%(wt/vol)BSA,在4℃下以1,500xg离心该板3分钟,并用多通道移液管除去上清液。 >
    25. 加入0.2ml PBS + 0.1%(wt/vol)BSA,并用多通道移液管混合样品
    26. 再次旋转板,并再次用PBS + 0.1%(wt/vol)BSA重复洗涤 将每个样品重悬在0.2ml PBS中,并将板保持在冰上,直到通过流式细胞术测量。在同一天测量非固定状态的样品。  

数据分析

注意:在含有吞噬体的PNS已经标记OVA和LAMP-1之后,通过流式细胞器细胞计数法分析以确定抗原降解的动力学(在给定时间点的吞噬体OVA的水平)和溶酶体标记物(例如,LAMP-1)的获得。由于含微球体吞噬体的物理性质和特定大小,可以应用门控策略以同时测量这些参数。

  1. 通过多色流式细胞术测量样品并应用以下门控策略:
    1. 通过单独测量OVA耦合珠确定PNS中珠和吞噬体的总体,并对样品应用类似的前向散射(FSC)和侧向散射(SSC)设置。在单个粒子群上设置一个门(图2A)。
    2. 从随后的分析中排除表面结合的珠(在手动裂解前标记的)。只包括吞噬体,通过应用第二个门(图2B),抗兔Alexa 568检测到的OVA为阴性。
    3. 门控群体包含单个吞噬体,其可以通过绘制OVA的直方图(通过抗兔DyLight 633检测)和LAMP-1的直方图,同时分析(图2C)或单独(图2D),随时间分析OVA降解和LAMP- 1(通过链霉亲和素Alexa Fluor 488检测) 注意:根据应用的抗体标记荧光团和使用的流式细胞仪,可能需要在不同通道之间进行补偿。
  2. 使用FlowJo软件(FlowJo,LLC。)进行流式细胞术数据的分析。使用Prism 6软件(GraphPad Software,Inc。)进行统计分析。每个单个实验需要通过足够数量的独立实验(至少一式三份)重复,以从流式细胞器细胞计数分析中得出结论。图2所示的数据代表至少三个独立实验

    图2. DC吞噬体的所应用的流式细胞器细胞计数分析的门控策略。使LPS处理的BMDC在16℃下内化OVA包被的珠,并孵育不同的追踪期(0分钟,60分钟,120分钟),以允许吞噬体成熟。表面结合的,非内化的珠子用抗OVA和Alexa 568偶联的二抗染色。随后,将细胞机械裂解以释放包括含有珠的吞噬体的核上清液(PNS)。 PNS对于OVA(通过DyLight 633偶联的第二抗体检测)和LAMP-1(通过Alexa 488偶联的第二抗体检测)染色,并通过流式细胞术同时分析。 A.使用标记的PNS的前向散射(FSC)和侧向散射(SSC)设置,将第一个门设置在单个珠和单个含珠的吞噬体上。 B.在第二个门中,从含珠的吞噬体中分离非内化珠(对Alexa 568是阳性的)(Alexa 568阴性)。 C.通过在成熟的OVA(Dylight 633) - 阴性和LAMP-1(Alexa 488)阳性群体上应用第三个门,同时分析噬菌粒以降解OVA和获得LAMP-1。 D.或者,吞噬体成熟的这两个参数也可以单独分析和随时间量化

笔记

  1. BMDC由含有GM-CSF的培养基中的分离的鼠骨髓祖细胞产生9天。在实验当天,CD11c的细胞表面表达应始终超过85%
  2. 结果的重现性高度依赖于机械破碎和细胞裂解期间的同质条件。始终通过台盼蓝染色控制细胞裂解的成功。另一个因素是使用的细胞的吞噬效率。始终计数您的样品的细胞数量,以应用相当的珠 - 细胞比率珠收购。不要超过这个比例,因为大量的内化颗粒会诱导细胞死亡,并影响流式细胞仪测量的参数。
  3. 虽然OVA耦合颗粒在这里用作吞噬模型系统,其他配体可以应用在珠偶联期间调查受体 - 配体相互作用对吞噬体成熟的影响。一些例子在别处公开(Hoffmann等人,2010; Hoffmann等人,2012)。

食谱

  1. 内部化媒介
    CO 2独立介质
    1x glutamax补充剂
  2. BMDC培养基
    IMDM
    10%(vol/vol)FBS
    50μMβ-巯基乙醇 100 IU/ml青霉素
    100μg/ml链霉素 来自J558浆细胞瘤细胞的10%(vol/vol)上清液,其用作GM-CSF来源(Winzler等人,1997)
  3. 均匀化缓冲液
    3mM咪唑,pH7.4 250mM蔗糖 2mM PMSF
    1x蛋白酶抑制剂混合物
    2 mM DTT

致谢

这项工作由法国国家研究机构通过"未来投资"计划(法国生物成像,ANR-10-INSB-04),ANR-11-LABX-0043和CelTisPhyBio Labex(N-ANR- 10-LBX-0038),IDEX PSL的一部分(ANR-10-IDEX-0001-02 PSL)。我们感谢欧洲研究委员会(2013-AdG No.340046 DCBIOX),La Ligue Nationale contrele Cancer(EL2014.LNCC/SA),Fonds Wetenschappelijk Onderzoek(FWO; 1526615N; 11W8415N)的资金支持, EMBO长期奖学金(ALTF 883-2011)以及Fondation RechercheMédicale奖学金(SPF20101221176)和omics @ VIB课程(由Marie Curie FP7 People Cofund共同资助)。
这里描述的协议是基于我们实验室以前发表的协议(Savina等人,2010),其被进一步开发以测量树突状细胞和巨噬细胞中的不同吞噬体成熟参数。

参考文献

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引用:Hoffmann, E., Pauwels, A., Alloatti, A., Kotsias, F. and Amigorena, S. (2016). Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry. Bio-protocol 6(22): e2014. DOI: 10.21769/BioProtoc.2014.
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