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Jul 2021

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Flow Cytometric Characterization of Macrophages Infected in vitro with Salmonella enterica Serovar Typhimurium Expressing Red Fluorescent Protein
表达红色荧光蛋白的肠沙门氏菌血清鼠伤寒菌体外感染巨噬细胞的流式细胞术研究   

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Abstract

Macrophages are important for host defense against intracellular pathogens like Salmonella and can be differentiated into two major subtypes. M1 macrophages, which are pro-inflammatory and induce antimicrobial immune effector mechanisms, including the expression of inducible nitric oxide synthase (iNOS), and M2 macrophages, which exert anti-inflammatory functions and express arginase 1 (ARG1). Through the process of phagocytosis, macrophages contain, engulf, and eliminate bacteria. Therefore, they are one of the first lines of defense against Salmonella. Infection with Salmonella leads to gastrointestinal disorders and systemic infection, termed typhoid fever. For further characterization of infection pathways, we established an in vitro model where macrophages are infected with the mouse Salmonella typhi correlate Salmonella enterica serovar Typhimurium (S.tm), which additionally expresses red fluorescent protein (RFP). This allows us to clearly characterize macrophages that phagocytosed the bacteria, using multi-color flow cytometry.


In this protocol, we focus on the in vitro characterization of pro- and anti-inflammatory macrophages displaying red fluorescent protein-expressing Salmonella enterica serovar Typhimurium, by multi-color flow cytometry.

Keywords: Salmonella Typhimurium (鼠伤寒沙门氏菌), Macrophages (巨噬细胞), Infection Control (感染控制), Arginase 1 (精氨酸酶 1), Inducible Nitric Oxide Synthase (诱导型一氧化氮合酶)

Background

The intracellular Gram-negative bacterium Salmonella typhi can cause severe and often life-threatening disease in humans. Globally, approximately 200,000 deaths are caused by the bacterium every year. The intracellular pathogen is ingested through contaminated food, and transmitted from person to person. The mouse correlate of human Salmonella typhi is Salmonella enterica serovar Typhimurium (S.tm). The intracellular bacteria are phagocytosed by macrophages and are able to evade antimicrobial defense by inhibiting fusion of lysosome and phagosome. Therefore, S.tm is able to survive and replicate inside the host (Buchmeier and Heffron, 1991; Navarre et al., 2010; Lahiri et al., 2010; Mastroeni and Grant, 2011; Bhutta et al., 2018).


In general, the immune system is divided into cells of the innate immune system (monocytes, macrophages, dendritic cells, and natural killer cells), and the acquired immune system (B lymphocytes, and T lymphocytes). Macrophages are phagocytic cells of the innate immune system, and one of the body's first defense mechanisms, when a pathogen crosses the host’s skin barrier. They can be classified into two types: (I) M1 pro-inflammatory macrophages, which are responsible for killing bacterial and viral pathogens, and express inducible nitric oxide synthase (iNOS), and (II) M2 macrophages, which support the wound healing process, and have an anti-inflammatory effect. Importantly, M2 macrophages express the enzyme arginase 1 (ARG1) (Mosser and Edwards, 2008; Mills, 2012; Weiss and Schaible, 2015; Gordon and Martinez-Pomares, 2017; Murray, 2017; Hannemann et al., 2019).


The cytosolic enzyme ARG1 is primarily expressed in liver tissue. During infection, ARG1 upregulation promotes pathogen survival in macrophages, by hydroxylating l-arginine to urea and ornithine, which therefore lowers l-arginine levels for the synthesis of nitric oxide (NO) by iNOS. A fully functional iNOS needs an adequate l-arginine supply to produce NO that kills pathogens. Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) are potent inducers of iNOS (Nairz et al., 2013; Bogdan, 2015; Brigo et al., 2021). ARG1 is induced by interleukin 4 (IL-4), which is produced by type 2 T helper cells. TNFα and IFNγ inhibit the transcription of ARG1, by interfering with IL-4–induced chromatin remodeling (Ostuni and Natoli, 2011; Schleicher et al., 2016; Piccolo et al., 2017). Additionally, several studies show that increased ARG1 activity is associated with an increased concentration of various pathogens, such as Streptococcus pneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis, or Toxoplasma gondii (Iniesta et al., 2005; El Kasmi et al., 2008; De Muylder et al., 2013; Schleicher et al., 2016; Paduch et al., 2019). However, it has been reported that deletion or pharmacological inhibition of ARG1 does not lead to a better control of Salmonella infection in macrophages, or in mice, in a septicemia model (Brigo et al., 2021).


Current treatment against invasive salmonellosis with antibiotics has become more difficult, due to resistance against conventional antibiotics. Therefore, the identification of new mechanisms to better understand the host-pathogen interplay in Salmonella infection, and the detailed characterization of the cells involved in the defense against this infection are urgently needed. Herein, we describe a method that identifies pro- and anti-inflammatory bone marrow-derived macrophage subtypes, during an infection with Salmonella enterica serovar Typhimurium. Furthermore, Salmonella expressing red fluorescent protein allows the visualization of phagocytosed Salmonella in these subtypes. This method supports further research on the involvement of pro- and anti-inflammatory macrophages in the defense against Salmonella.

Materials and Reagents

  1. 250 mL Erlenmeyer flask (Stoelzle Medical, catalog number: 21226368000)

  2. 0.5 mL Eppendorf tubes (Eppendorf, catalog number: 0030121.023)

  3. Disposable cuvette (BRAND, catalog number: 759015)

  4. 1.5 mL Eppendorf tubes (Eppendorf, catalog number: 0030120.086)

  5. Cell scraper (Sarstedt, catalog number: 83.3951)

  6. 15 mL Polypropylene conical tube (Falcon, catalog number: 352096)

  7. Cell strainer 40 µm (Falcon, catalog number: 352360)

  8. 96-well BRAND plate (Life Science Products, catalog number: 781602)

  9. 5 mL disposable syringe (BD, catalog number: 309050)

  10. Luna cell counting slides (Biocat, catalog number: L201B1C3GB)

  11. 6-cm dish (TTP, catalog number: 93060)

  12. Salmonella enterica serovar Typhimurium SL1344 expressing red fluorescent protein (RFP) (Birmingham et al., 2006; Wu et al., 2017)

  13. Lysogeny broth (LB Broth) Lennox (Roth, catalog number: X964.2)

  14. Glycerol (Sigma, catalog number: G5516-100ML)

  15. Phosphate buffer saline (PBS; Lonza, catalog number: 17-515 F)

  16. Agar-Agar Kobe I (Roth, catalog number: 5210.3)

  17. CASY Cup (OMNI Life Science, catalog number: 5651794)

  18. CASY Ton Buffer (OMNI Life Science, catalog number: 5651808)

  19. Aqua bidest (Fresenius Kabi, catalog number: 16.231)

  20. Gentamicin (Gibco, catalog number: 15750-037)

  21. L-glutamine (Lonza, catalog number: BE17-605E)

  22. Dulbecco′s Modified Eagle′s Medium (DMEM, Pan BiotechTM, catalog number: P04-01500)

  23. Fetal bovine serum (FBS, Pan BiotechTM, catalog number: P30-3031)

  24. BV650 anti-mouse/human CD11b (BioLegend, catalog number: 101239)

  25. APC-R700 rat anti-mouse CD45 (BDHorizonTM, catalog number: 565478)

  26. BV421 rat anti-mouse F4/80 (BD Biosciences; catalog number: 565411)

  27. PerCP-eFluorTM 710 anti-mouse Ly6G (Invitrogen; catalog number: 46-9668-82)

  28. FITC anti-mouse CD3 (BioLegend, catalog number: 100204)

  29. FITC anti-mouse CD19 (ImmunoTools, catalog number: 22220193S)

  30. FITC anti-mouse CD49b (BioLegend, catalog number: 103503)

  31. PE-Cyanine7 anti-mouse iNOS (Invitrogen; catalog number: 25-5920-80)

  32. APC anti-mouse ARG1 (Invitrogen, catalog number: 17-3697-82)

  33. BD Cytofix/CytopermTM (BD Biosciences, catalog number: 51-2091K7)

  34. BD PermWashTM (BD Biosciences, catalog number: 51-2090K7)

  35. 50 mL Polypropylene conical tube (Falcon, catalog number: 352070)

  36. Ketamine (Livisto, catalog number: 6680219)

  37. Xylazine (Animedica, catalog number: 7630517)

  38. Omnican F syringes (Braun, catalog number: 91615025)

  39. Disposable hypodermic needle 100 Sterican R (Braun, catalog number: 4657519)

  40. Pen-Strep (Lonza, catalog number: DE17-602E)

  41. Erythrocyte lysis buffer (R&D, catalog number: WL2000)

  42. Acridine Orange/propidium iodide stain (Biocat, catalog number: F23001)

  43. LB medium (see Recipes)

  44. LB-medium with 30% Glycerol (see Recipes)

Equipment

  1. Laminar Flow Cabinet; EuroClone Safe Mate Eco 1.2 (Politakis Laborgeräte, catalog number: EN 12 469)

  2. Shaking incubator (VWR, catalog number: GFL 3031)

  3. Heraeus® HERAcell® CO2 Incubator (Thermo Fisher Scientific, catalog number: 3615-45)

  4. Photometer (Eppendorf, BioPhotometer D30, catalog number: 6133000001)

  5. Centrifuge (Hettich Micro 200R and Rotanta 460R, catalog number: Z652113, Z623520)

  6. CASY TT counting system (OMNI Life Science, catalog number: TT-20A-2571)

  7. CytoFLEX S V4-B4-R2-I2 Flow Cytometer (13 detectors, 4 lasers, Beckman Coulter, catalog number: C01161)

  8. LUNA Automated Cell Counter (Biocat, L10001-LG)

Software

  1. FlowJo v10.7.0 (BD Biosciences, https://www.flowjo.com/solutions/flowjo)

    Note: Any software package for analyzing flow cytometry data can be used with this protocol.

Procedure

  1. Preparation of Salmonella Typhimurium (S.tm) stock

    1. Take an aliquot of Salmonella enterica serovar Typhimurium SL1344 expressing red fluorescent protein (RFP) from -20°C storage.

    2. Thaw the aliquot at room temperature.

    3. Pipette 10 µL of S.tm into 10 mL of LB-medium in a 250-mL Erlenmeyer flask.

    4. Cover the top of the flask using tin foil.

    5. Incubate in a shaking incubator at 200 rpm and 37°C overnight.

    6. The following day, pipette 50 µL of the overnight culture into 10 mL of fresh LB-medium into a 250-mL Erlenmeyer flask.

    7. Discard the overnight culture of S.tm. Wash and sterilize the 250-mL Erlenmeyer flask.

    8. Cover the top of the flask using tin foil.

    9. Incubate the culture in a shaking incubator at 200 rpm and 37°C for 1–2 h.

    10. Calibrate a photometer, using 500 µL of LB-medium in a disposable cuvette as blank.

    11. Measure OD600, to check if S.tm reached 0.5.

      S.tm reaches the optimal logarithmic growth phase when OD600 is between 0.5–0.7.

      Note: If the OD600 value is below 0.5, continue the incubation of the culture in the 250-mL Erlenmeyer flask as described above, until an OD600 value of 0.5 is reached. Of note, S.tm density duplicates every 20 min. If the OD600 value is above 0.7, dilute the culture 1:1 with LB-medium, and incubate it in the 250-mL Erlenmeyer flask, until an OD600 value of 0.5.

    12. Transfer the culture into a 50-mL conical tube.

    13. Centrifuge the S.tm culture at 4,967 × g at room temperature for 5 min.

    14. Discard the supernatant.

    15. Resuspend the pellet in 1 mL of freshly prepared LB-medium + 30% glycerol.

    16. Prepare aliquots of 50 µL in 0.5-mL Eppendorf tubes, and store them at -20°C.


  2. Culture of S.tm to the optimal growth phase

    1. Thaw one aliquot of the S.tm stock.

    2. Pipette 10 µL of this aliquot into 10 mL of LB-medium in a 250-mL Erlenmeyer flask, and cover the top of the flask using tin foil.

    3. Incubate in a shaking incubator at 200 rpm and 37°C overnight.

    4. The following day, pipette 50 µL of this overnight culture into 10 mL of LB-medium in a 250-mL Erlenmeyer flask, and cover the top of the flask using tin foil.

    5. Discard the overnight culture of S.tm. Wash and sterilize the 250 mL Erlenmeyer flask.

    6. Incubate in a shaking incubator at 200 rpm and 37°C for 1–2 h.

    7. Calibrate a photometer, using 500 µL of LB medium in a disposable cuvette as blank.

    8. Measure OD600 to check if S.tm reached 0.5, which is equivalent to the optimal logarithmic growth phase.

      Note: If the OD600 value is below 0.5, continue the incubation of the culture in the 250-mL Erlenmeyer flask as described above, until an OD600 value of 0.5 is reached. Of note, S.tm density duplicates every 20 min. If the OD600 value is above 0.7, dilute the culture 1:1 with LB-medium, and incubate it in the 250-mL Erlenmeyer flask, until an OD600 value of 0.5.


  3. Counting viable S.tm using a Casy counting system

    1. Use the 45-µm capillary.

    2. Measure the background by placing a new Casy cup with 10 mL of fresh Casy ton buffer under the measuring unit.

    3. Select the program for background measurement (Table 1 Background Measurement).

    4. Measure the background. This should be below 30 counts and 1 µm in size. Otherwise, wash the system.

    5. Prepare a new Casy cup with 10 mL of Casy ton buffer, and add 5 µL of S.tm OD600 0.5.

    6. Shake gently.

    7. Place the sample under the measuring unit.

    8. Select the program for measuring between 1–3 µm (Table 1S.tm Measurement).

    9. Measure.

    10. Click next, to get the number of viable counts/mL = viable S.tm /mL.

      Note: Viable counts from a freshly prepared S.tm culture with an OD600 of 0.5 should be between 2.5 × 108–3 × 108 viable counts/mL.

    11. After the measurement is finished, remove the sample cup, and add a fresh Casy cup with 10 mL of Casy ton buffer.

    12. Perform Casy Clean up to five times.

    13. Select the program for washing (Table 1 Washing Program).

    14. After the washing is completed, check the background again.

    15. If the background is below 30, the Casy counting system can be turned off. Otherwise, continue the washing.


      Table 1. Programs CASY TT Counting system

      Background

      Measurement

      Measurement

      of S. Typhimurium

      Washing Program
      Capillary 45 µm X-Axis: 5 µm 45 µm X-Axis: 3 µm 45 µm X-Axis: 5 µm
      Sample Volume 200 µL Cycles: 1 200 µL Cycles: 3 200 µL Cycles: 10
      Dilution 1.001e+00 2.001e+03 1.001e+00
      Y-Axis Auto Auto Auto
      Eval.Cursor 1.00–4.89 µm 0.75–2.93 µm 0.00–5.00 µm
      Norm. Cursor 0.5–4.89 µm 0.3–2.93 µm 0.00–5.00 µm
      %Calculation %Via Debris: On %Via Debris: On %Via Debris: On
      Aggr. Correct: Auto Auto Auto P
      Interface Par P.Feed: On Par P.Feed: On Par P.Feed: On
      Print Mode Manual Graphic: On Manual Graphic: On Manual Graphic: On


  4. Preparation of bone marrow-derived macrophages (BMDM)

    Note: Preparation of BMDM has been described by Zanoni et al. (2012; doi:10.21769/BioProtoc.225.).

    A video demonstrating the procedure can be found at: https://www.jove.com/de/v/52347/isolation-intravenous-injection-murine-bone-marrow-derived.

    A schematic illustration of the generation of bone marrow-derived macrophages and infection with S.tm is shown in Figure 1.



    Figure 1. Schematic representation of the experimental setup, showing the generation of bone marrow-derived macrophages and infection with S.tm.

    The different morphological shapes of uninfected and infected BMDM are visualized in a ScanR Imaging Platform (pictures kindly provided by Demetz E.).


    1. Anesthetize a wildtype C57Bl/6N mouse, by intraperitoneally injecting 50 µL of 100 mg/kg Ketamine + 10 mg/kg Xylazine.

      Note: A video demonstrating the procedure in general can be found at Intraperitoneal Injection in the Mouse: https://researchanimaltraining.com/articles/intraperitoneal-injection-in-the-mouse/.

    2. Perform euthanization of the deeply anesthetized mouse by cervical dislocation. Therefore, place a large tweezer behind the base of the anesthetized mouse's skull, and pull sharply back on the tail at a 45° angle.

    3. Fixate the animal on a Styrofoam panel, and spray the surface of the animal with 75% alcohol.

    4. Remove skin and muscle tissue from one leg, by cutting upwards from the heel with sterile scissors.

    5. Cut around the femur head.

    6. Cut in the middle of the knee joint. Be careful not to damage the bones.

    7. Cut the ankle joint.

    8. Remove excess muscles with tissue paper.

    9. Pull on the upper leg, to remove the femur head from the hip joint.

    10. Place the bones into PBS containing 1% penicillin and 1% streptomycin on ice.

    11. Move to a laminar flow hood, and perform all steps on ice.

    12. Open the ends of the bones, by cutting with a pair of sterile scissors.

    13. Place a 40-µm cell strainer on a 50-mL Falcon-tube.

    14. Flush out the bone marrow:

      1. Use a disposable hypodermic needle and a 5-mL syringe.

      2. Fill the syringe with PBS containing 1% penicillin and 1% streptomycin.

      3. Place a needle on one end of the opened bone.

      4. Flush the bone marrow out onto the 40-µm cell strainer.

      5. Repeat flushing of the bone, until it is completely white.

    15. Wash the cell strainer with 10 mL of PBS containing 1% penicillin and 1% streptomycin.

    16. Using the plunger of the syringe, strain the cells through the cell strainer.

    17. Wash the cell strainer with 10 mL of PBS containing 1% penicillin and 1% streptomycin.

    18. Centrifuge the 50-mL conical tube containing your flushed bone marrow at 300 × g and 4°C for 5 min.

    19. Dilute Erythrocyte Lysis Buffer 1:10 with double distilled water

    20. Discard the supernatant.

    21. Resuspend the pellet in 2 mL of diluted Erythrocyte Lysis Buffer.

    22. Incubate at room temperature for 3 min.

    23. Add 15 mL of PBS containing 1% penicillin and 1% streptomycin on top of the Erythrocyte Lysis Buffer.

    24. Centrifuge the 50-mL conical tube containing your flushed bone marrow at 300 × g and 4°C for 5 min.

    25. Discard the supernatant.

    26. Resuspend the pellet in 15 mL of PBS containing 1% penicillin and 1% streptomycin.

    27. Repeat steps 24–26.

    28. Centrifuge the 50-mL conical tube again, and discard the supernatant.

    29. Resuspend the cell pellet in 45 mL of DMEM media supplemented with 10% FBS, 1% L-glutamine, 1% penicillin, 1% streptomycin, and 50 ng/mL recombinant murine M-CSF.

    30. Pipette 15 mL of the cell suspension into each of three 20-cm Falcon dishes.

    31. Change the medium every second day.

    32. On day 5, cells can be harvested (Procedure E).


  5. Harvesting and counting of cells

    1. Remove the culture media from the cell culture dishes.

    2. Wash the cells twice with 10 mL of PBS.

    3. Add 8 mL of DMEM medium supplemented with 10% FBS and 1% L-glutamine.

    4. Scrape the cells using a disposable cell scraper.

    5. Wash the dishes with another 2 mL of DMEM medium supplemented with 10% FBS and 1% L-glutamine.

    6. Transfer the cells into a 50-mL conical tube.

    7. Close the tube, and invert the cells 2–3 times.

    8. Place 9 µL of the cell suspension in a 0.5-µL Eppendorf tube.

    9. Mix 1 µL of Acridine Orange/ Propidium Iodide stain solution with the cell aliquot.

    10. Place 10 µL of the mixture into a Luna cell counting slide.

    11. Count the cells using the LUNA-FL fluorescent and bright field automated cell counter.

      Note: Approximately 4.5 × 107 cells are obtained from one mouse, after isolating and culturing the bone marrow of both hind legs.

    12. Seed the cells in 6-cm dishes, at a density of 1.5 × 106 cells/mL in DMEM medium supplemented with 10% FBS and 1% L-glutamine.

    13. Seed four additional dishes for fluorescent minus one (FMO) control (see step G3).

    14. Let the cells settle in a cell incubator overnight.


  6. In vitro infection of bone marrow-derived macrophages with S.tm

    1. Infect BMDM with S.tm at a multiplicity of infection 10 (MOI 10). Therefore, add 10 times more S.tm than cells.

      Note: Unused S.tm culture with OD600 of 0.5 can be used for preparing new S.tm aliquots (Procedure A), or be discarded.

    2. Incubate the cells in a cell incubator (5% CO2, 37°C) for 1 h.

    3. Remove the medium containing non-phagocytosed S.tm.

    4. Wash the cells twice with 1 mL of PBS + 25 µg/mL gentamicin.

    5. Add 1 mL of DMEM medium supplemented with 10% FBS, 1% L-glutamine, and 25 µg/mL gentamicin.

    6. Incubate the cells in a cell incubator for 4 h.


  7. Flow Cytometry staining of cultured BMDM

    1. Extracellular staining

      1. Harvest the BMDM by scraping in culture medium, using a disposable cell scraper.

      2. Transfer the cells to a 15-mL Falcon tube, and pellet by centrifugation at 300 × g and 4°C for 5 min.

      3. Discard the supernatant.

      4. Resuspend the pellet in 1 mL of PBS, and transfer it into an 1.5-µL Eppendorf tube.

      5. Pellet the cells by short spinning at 10,860 × g and 4°C for 30 s.

      6. Discard the supernatant.

      7. Resuspend the pellet in 50 µL of surface antibody mix (all antibodies 1:200 in PBS; Table 2).


        Table 2. Antibodies for flow cytometry – extracellular stain.

        Antibody Clone Company Catalog number expressed on
        CD3 FITC 17.A2 BioLegend 100204 T cells
        CD19 FITC PeCa1 ImmunoTools 22220193S B cells
        CD49b FITC HMa2 BioLegend 103503 NK cells
        CD11b BV650 M170 BioLegend 101239 neutrophils, monocytes
        CD45 APC-R700 30-F11 BD Horizon 565478 leukocytes
        F4/80 BV421 T45-2342 BD Horizon 565411 macrophages
        Ly6G PerCP-eFlour 710 1AB-Ly6g Invitrogen 46-9668-82 neutrophils

        Note: To be sure that the bone marrow-derived macrophages are not contaminated by T cells, B cells, or NK cells, antibodies against CD3, CD19, and CD49b are added to the flow cytometry panel. All these antibodies are labeled with FITC; therefore, all FITC+ cells can be excluded in the gating strategy (Figure 2). The typical cell distribution of a BMDM culture (uninfected and infected with S.tm) is shown in Table 4.


      8. Incubate in the dark at 4°C for 10 min.

      9. Wash with 500 µL of PBS.

      10. Pellet the cells by short spinning (10,860 × g, 4°C, 30 s).

      11. Discard the supernatant.

      12. Resuspend the pellet in 100 µL of Cytofix/CytoPermTM Buffer, to permeabilize and fix the cells.

      13. Incubate in the dark at 4°C for 20 min.

      14. Dilute the PermWashTM Buffer 1:10 with Aqua bidest.

      15. Add 500 µL of the diluted PermWashTM Buffer on top of the 100 µL of Cytofix/CytoPermTM Buffer.

      16. Pellet the cells by short spinning (10,860 × g, 4°C, 30 s).

      17. Discard the supernatant.

    2. Intracellular stain

      1. Prepare the intracellular antibody mix in diluted PermWashTM Buffer (iNOS 1:100, and ARG1 1:100; Table 3).


        Table 3. Antibodies for flow cytometry – intracellular stain.

        Antibody Clone Company Catalog number expressed on
        iNOS PE-Cyanine7 CXNFT Invitrogen 25-5920-80 pro-inflammatory macrophages
        ARG1 APC A1exF5 Invitrogen 17-3697-82 anti-inflammatory macrophages


      2. Resuspend the samples in 50 µL of intracellular antibody mix.

      3. Incubate the samples protected from light at room temperature for 45 min.

      4. Wash the cells once with 500 µL of diluted PermWashTM. Pellet the cells by short spinning (10,860 × g, 4°C, 30 s).

      5. Discard the supernatant, and resuspend the pellets in 200 µL of PBS.

      6. Transfer the samples into a flat bottom 96-well plate, via a 40-µm strainer.

      7. Analyze directly in a flow cytometry device.

    3. Fluorescent minus one (FMO) control

      1. Perform extracellular staining of BMDM, as described in step G1, in the additionally seeded uninfected and infected BMDM samples (see step E13).

      2. Discard the supernatant after washing with diluted PermWashTM Buffer on top of the 100 µL of Cytofix/CytoPermTM Buffer.

      3. Resuspend one uninfected and one infected BMDM cell pellets in the intracellular antibody mix with only the antibody against ARG1. Resuspend the other uninfected and infected BMDM cell pellets in the intracellular antibody mix with only the antibody against iNOS.

      4. Incubate the samples protected from light at room temperature for 45 min.

      5. Wash the cells once with 500 µL of diluted PermWashTM.

      6. Pellet the cells by short spinning (10860 × g, 4°C, 30 s).

      7. Discard the supernatant, and resuspend the pellets in 200 µL of PBS.

      8. Transfer the samples into a flat bottom 96-well plate, via a 40-µm strainer.

      9. Analyze directly in a flow cytometry device.



    Figure 2. Gating strategy for the infected bone marrow-derived macrophages – upper panel, and the uninfected bone marrow-derived macrophages – lower panel.

    After exclusion of doublets, the leukocytes are described as CD45+. T-cells, B-cells, and NK cells are excluded (CD3CD19CD49b), and macrophages are gated as Ly6GCD11b+F4/80+. Depending on the research question, there is the possibility to characterize pro-inflammatory macrophages as iNOS-expressing cells, and anti-inflammatory macrophages as ARG1–expressing cells. Furthermore, macrophages containing S.tm are characterized by expression of the red fluorescent protein (RFP) in the PE channel.

Data analysis

The FlowJo software was used to analyze the data. The gating strategy is described in Figure 2.

In BMDM generated from C57BL/6N mice that were either left uninfected or were infected with S.tm, and afterwards further incubated for 4h, typical values for analyzed cell subsets are depicted in Table 4.


Table 4. Typical cell distribution of a BMDM culture.

Cell type Gating Mean ± SEM Uninfected Mean ± SEM Infected
Cells FSC-A to SSC-A 94.7 ± 2.1 95.2 ± 2.1
Single Cells FSC-A to FSC-H 90.0 ± 2.7 91.7 ± 2.2
Leukocytes CD45 to FSC-A 98.6 ± 1.3 99.5 ± 0.3
FITC- negative cells FITC (CD3, CD19, CD49b) to FSC-A 98.6 ± 0.7 97.7 ± 0.6
Monocytes (Ly6G-) Ly6G to FSC-A 98.8 ± 0.7 98.4 ± 0.5
Macrophages F4/80 to CD11b 98.0 ± 0.9 98.8 ± 1.1
Anti-inflammatory macrophages ARG1 to FSC-A 1.8 ± 0.5 1.4 ± 0.3
Pro-inflammatory macrophages iNOS to FSC-A 4.2 ± 2.3 77.7 ± 3.8
Salmonella containing macrophages STR to FSC-A 0 24.7 ± 1.78

Troubleshooting

  1. Cultivation of S.tm (see Procedures A and B)

    Salmonella grow best at 37°C, and need oxygen for optimal growth. Therefore, they should be cultivated in an Erlenmeyer flask which is covered with tin foil. The temperature of 37°C must be strictly ensured.


  2. Preparation of BMDM (see Procedure D)

    For the generation of bone marrow-derived macrophages, it is important to work in a sterile manner. Cells should only be handled in a laminar flow cabinet. It is important to isolate the entire tibiae and femura, and to cut them open only under sterile conditions, using autoclaved scissors and tweezers.


  3. Harvesting, counting, and seeding of BMDM (see Procedure E)

    Harvesting of BMDM by scraping needs to be performed rather gently, to obtain high cell viability. We recommend scraping away from one’s body, exerting not much force on the disposable cell scraper.

    Cells are seeded for infection in antibiotic free media. Therefore, the day before seeding, it is recommended to incubate 5 mL of the antibiotic free medium in a cell culture dish placed in a cell incubator overnight, to be sure that the medium is not contaminated by bacteria.


  4. Infection of BMDM (see Procedure F)

    After infection, it is necessary to thoroughly wash away the unphagocytosed S.tm. It is important to remove the medium completely, and wash the cells at least twice with PBS + gentamicin. A microscope can be used to see whether S.tm are still present in the culture. If yes, washing steps should be repeated.

    It is important to add gentamicin to the PBS for washing, and to the DMEM for further incubation. Gentamicin inhibits the proliferation of S.tm, by blocking protein biosynthesis.

Recipes

  1. LB medium

    a.d. with

    2% LB-Broth

    Autoclave (at 121°C for 20 min, and at 50°C for 10 min)

  2. LB medium with 30% Glycerol

    Add 300 µL of Glycerol to 700 µL of LB medium

Acknowledgments

G.W. is supported by grants from the Christian Doppler Society and an ERA-NET grant by the FWF (EPICROSS, I-3321), and N.B. was supported by the FWF doctoral college project W1253 HOROS.

Salmonella Typhimurium expressing red fluorescent protein were a kind gift from Prof. Dr. Dirk Bumann (University of Basel, Switzerland). This protocol was adapted and modified after Fritsche et al. (2008).

Competing interests

The authors declare no conflicts of interest.

References

  1. Bhutta, Z. A., Gaffey, M. F., Crump, J. A., Steele, D., Breiman, R. F., Mintz, E. D., Black, R. E., Luby, S. P. and Levine, M. M. (2018). Typhoid Fever: Way Forward. Am J Trop Med Hyg 99(3_Suppl): 89-96.
  2. Birmingham, C. L., Smith, A. C., Bakowski, M. A., Yoshimori, T. and Brumell, J. H. (2006). Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole. J Biol Chem 281(16): 11374-11383.
  3. Bogdan, C. (2015). Nitric oxide synthase in innate and adaptive immunity: an update. Trends Immunol 36(3): 161-178.
  4. Brigo, N., Pfeifhofer-Obermair, C., Tymoszuk, P., Demetz, E., Engl, S., Barros-Pinkelnig, M., Dichtl, S., Fischer, C., Valente De Souza, L., Petzer, V., et al. (2021). Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection.Cells 10(7).
  5. Buchmeier, N. A. and Heffron, F. (1991). Inhibition of macrophage phagosome-lysosome fusion by Salmonella typhimurium.Infect Immun 59(7): 2232-2238.
  6. De Muylder, G., Daulouede, S., Lecordier, L., Uzureau, P., Morias, Y., Van Den Abbeele, J., Caljon, G., Herin, M., Holzmuller, P., Semballa, S., et al. (2013). A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity. PLoS Pathog 9(10): e1003731.
  7. El Kasmi, K. C., Qualls, J. E., Pesce, J. T., Smith, A. M., Thompson, R. W., Henao-Tamayo, M., Basaraba, R. J., Konig, T., Schleicher, U., Koo, M. S., et al. (2008). Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens.Nat Immunol 9(12): 1399-1406.
  8. Fritsche, G., Nairz, M., Werner, E. R., Barton, H. C. and Weiss, G. (2008). Nramp1-functionality increases iNOS expression via repression of IL-10 formation.Eur J Immunol 38(11): 3060-3067.
  9. Gordon, S. and Martinez-Pomares, L. (2017). Physiological roles of macrophages. Pflugers Arch 469(3-4): 365-374.
  10. Hannemann, N., Cao, S., Eriksson, D., Schnelzer, A., Jordan, J., Eberhardt, M., Schleicher, U., Rech, J., Ramming, A., Uebe, S., et al. (2019). Transcription factor Fra-1 targets arginase-1 to enhance macrophage-mediated inflammation in arthritis. J Clin Invest 129(7): 2669-2684.
  11. Iniesta, V., Carcelen, J., Molano, I., Peixoto, P. M., Redondo, E., Parra, P., Mangas, M., Monroy, I., Campo, M. L., Nieto, C. G., et al. (2005). Arginase I induction during Leishmania major infection mediates the development of disease. Infect Immun 73(9): 6085-6090.
  12. Lahiri, A., Lahiri, A., Iyer, N., Das, P. and Chakravortty, D. (2010). Visiting the cell biology of Salmonella infection.Microbes Infect 12(11): 809-818.
  13. Mastroeni, P. and Grant, A. J. (2011). Spread of Salmonella enterica in the body during systemic infection: unravelling host and pathogen determinants. Expert Rev Mol Med 13: e12.
  14. Mills, C. D. (2012). M1 and M2 Macrophages: Oracles of Health and Disease.Crit Rev Immunol 32(6): 463-488.
  15. Mosser, D. M. and Edwards, J. P. (2008). Exploring the full spectrum of macrophage activation.Nat Rev Immunol 8(12): 958-969.
  16. Murray, P. J. (2017). Macrophage Polarization. Annu Rev Physiol 79: 541-566.
  17. Nairz, M., Schleicher, U., Schroll, A., Sonnweber, T., Theurl, I., Ludwiczek, S., Talasz, H., Brandacher, G., Moser, P. L., Muckenthaler, M. U., et al. (2013). Nitric oxide-mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection. J Exp Med 210(5): 855-873.
  18. Navarre, W. W., Zou, S. B., Roy, H., Xie, J. L., Savchenko, A., Singer, A., Edvokimova, E., Prost, L. R., Kumar, R., Ibba, M., et al. (2010). PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica. Mol Cell 39(2): 209-221.
  19. Ostuni, R. and Natoli, G. (2011). Transcriptional control of macrophage diversity and specialization.Eur J Immunol 41(9): 2486-2490.
  20. Paduch, K., Debus, A., Rai, B., Schleicher, U. and Bogdan, C. (2019). Resolution of Cutaneous Leishmaniasis and Persistence of Leishmania major in the Absence of Arginase 1. J Immunol 202(5): 1453-1464.
  21. Piccolo, V., Curina, A., Genua, M., Ghisletti, S., Simonatto, M., Sabo, A., Amati, B., Ostuni, R. and Natoli, G. (2017). Opposing macrophage polarization programs show extensive epigenomic and transcriptional cross-talk. Nat Immunol 18(5): 530-540.
  22. Schleicher, U., Paduch, K., Debus, A., Obermeyer, S., Konig, T., Kling, J. C., Ribechini, E., Dudziak, D., Mougiakakos, D., Murray, P. J., et al. (2016). TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection.Cell Rep 15(5): 1062-1075.
  23. Weiss, G. and Schaible, U. E. (2015). Macrophage defense mechanisms against intracellular bacteria.Immunol Rev 264(1): 182-203.
  24. Wu, A., Tymoszuk, P., Haschka, D., Heeke, S., Dichtl, S., Petzer, V., Seifert, M., Hilbe, R., Sopper, S., Talasz, H., et al. (2017). Salmonella Utilizes Zinc To Subvert Antimicrobial Host Defense of Macrophages via Modulation of NF-kappaB Signaling.Infect Immun 85(12).
  25. Zanoni, I., Ostuni, R. and Granucci, F. (2012). Generation of Mouse Bone Marrow-Derived Macrophages (BM-MFs). Bio-protocol 2(12): e225.

简介

[摘要]巨噬细胞对宿主防御沙门氏菌等细胞内病原体具有重要作用 并可分为两大亚型。 M1 巨噬细胞是促炎细胞,可诱导抗菌免疫效应机制,包括诱导型一氧化氮合酶 ( iNOS ) 的表达,以及 M2 巨噬细胞,它们发挥抗炎功能并表达精氨酸酶 1 (ARG1)。通过吞噬作用,巨噬细胞包含、吞噬和消灭细菌。因此,它们是抵御沙门氏菌的第一道防线之一。 感染沙门氏菌会导致胃肠道疾病和全身感染,称为伤寒。为了进一步表征感染途径,我们建立了一个体外模型,其中巨噬细胞感染小鼠伤寒沙门氏菌与鼠伤寒沙门氏菌 ( S. tm) 相关,后者还表达红色荧光蛋白 (RFP)。这使我们能够使用多色流式细胞术清楚地表征吞噬细菌的巨噬细胞。
在本协议中,我们专注于通过多色流式细胞仪对显示红色荧光蛋白的促炎和抗炎巨噬细胞进行体外表征。


[背景] 细胞内革兰氏阴性菌伤寒沙门氏菌可在人类中引起严重且经常危及生命的疾病。在全球范围内,每年约有 200,000 人死于这种细菌。细胞内病原体通过受污染的食物摄入,并在人与人之间传播。人伤寒沙门氏菌的小鼠相关物是鼠伤寒沙门氏菌血清型 ( S. tm)。细胞内细菌被巨噬细胞吞噬,并且能够通过抑制溶酶体和吞噬体的融合来逃避抗菌防御。因此, S. tm能够在宿主体内生存和复制(Buchmeier 和 Heffron,1991;Navarre等,2010;Lahiri等,2010;Mastroeni 和 Grant,2011;Bhutta等,2018) 。
一般来说,免疫系统分为先天免疫系统细胞(单核细胞、巨噬细胞、树突状细胞和自然杀伤细胞)和获得性免疫系统(B淋巴细胞和T淋巴细胞)。巨噬细胞是先天免疫系统的吞噬细胞,当病原体穿过宿主的皮肤屏障时,它是身体的第一个防御机制之一。它们可分为两种类型:(I) M1 促炎巨噬细胞,负责杀死细菌和病毒病原体,并表达诱导型一氧化氮合酶 ( iNOS ),以及 (II) M2 巨噬细胞,支持伤口愈合过程,并具有抗炎作用。重要的是,M2 巨噬细胞表达精氨酸酶 1 (ARG1) (Mosser 和 Edwards,2008;Mills,2012;Weiss 和 Schaible,2015;Gordon 和 Martinez-Pomares,2017;Murray,2017;Hannemann等人,2019) 。
胞质酶 ARG1 主要在肝组织中表达。在感染期间,ARG1 上调通过将 l-精氨酸羟基化为尿素和鸟氨酸来促进巨噬细胞中病原体的存活,从而降低iNOS合成一氧化氮 (NO) 的 l-精氨酸水平。功能齐全的iNOS需要充足的 l-精氨酸供应来产生杀死病原体的 NO。干扰素 γ (IFN ) 和肿瘤坏死因子 α (TNF ) 是iNOS的强效诱导剂 (Nairz等人,2013;Bogdan,2015;Brigo等人,2021) 。 ARG1 由 2 型 T 辅助细胞产生的白细胞介素 4 (IL-4) 诱导。 TNF和 IFN通过干扰 IL-4 诱导的染色质重塑来抑制 ARG1 的转录( Ostuni 和 Natoli,2011 ; Schleicher等人,2016 ; Piccolo等人,2017 ) 。此外,一些研究表明,ARG1 活性的增加与各种病原体的浓度增加有关,例如肺炎链球菌、牛分枝杆菌、结核分枝杆菌或刚地弓形虫 (Iniesta等人,2005;El Kasmi等人,2008;De Muylder等人,2013;Schleicher等人,2016;Paduch等人,2019) 。然而,据报道,在败血症模型中,ARG1 的缺失或药理学抑制不会导致更好地控制巨噬细胞或小鼠中的沙门氏菌感染( Brigo等人,2021 年) 。
由于对传统抗生素的耐药性,目前用抗生素治疗侵袭性沙门氏菌病变得更加困难。因此,迫切需要确定新机制以更好地了解沙门氏菌感染中的宿主-病原体相互作用,以及对参与防御这种感染的细胞进行详细表征。在这里,我们描述了一种在感染鼠伤寒沙门氏菌期间识别促炎和抗炎骨髓衍生巨噬细胞亚型的方法。此外,沙门氏菌 表达红色荧光蛋白允许在这些亚型中显示被吞噬的沙门氏菌。该方法支持进一步研究促炎和抗炎巨噬细胞参与防御沙门氏菌。

关键字:鼠伤寒沙门氏菌, 巨噬细胞, 感染控制, 精氨酸酶 1, 诱导型一氧化氮合酶



材料和试剂


1. 250 mL锥形瓶( Stoelzle Medical,目录号:21226368000)
2. 0.5 mL Eppendorf 管(Eppendorf,目录号: 0030121.023)
3. 一次性比色皿(BRAND,目录号: 759015)
4. 1.5 mL Eppendorf 管(Eppendorf,目录号: 0030120.086)
5. 细胞刮刀( Sarstedt ,目录号: 83.3951)
6. 15 mL聚丙烯锥形管(Falcon,目录号: 352096)
7. 细胞过滤器40 µm(Falcon,目录号:352360)
8. 96孔BRAND板(Life Science Products,目录号: 781602)
9. 5 mL一次性注射器(BD,目录号:309050)
10. Luna细胞计数载玻片( Biocat ,目录号:L201B1C3GB)
11. 6厘米培养皿(TTP,目录号:93060 )
12. 肠沙门氏菌血清型 鼠伤寒 SL1344 表达红色荧光蛋白 (RFP) (Birmingham et al. , 2006 ; Wu et al. , 2017)
13. 溶原肉汤(LB Broth)Lennox(Roth,目录号:X964.2)
14. 甘油(Sigma,目录号:G5516-100ML)
15. 磷酸盐缓冲盐水(PBS;Lonza,目录号: 17-515 F)
16. Agar-Agar Kobe I(Roth,目录号: 5210.3)
17. CASY Cup(OMNI Life Science,目录号: 5651794)
18. CASY Ton Buffer(OMNI Life Science,目录号: 5651808)
19. Aqua bidest (Fresenius Kabi ,目录号: 16.231)
20. 庆大霉素(Gibco,目录号: 15750-037)
21. L-谷氨酰胺(Lonza,目录号: BE17-605E)
22. Dulbecco 改良 Eagle 培养基(DMEM,Pan Biotech TM ,目录号: P04-01500)
23. 胎牛血清(FBS,Pan Biotech TM ,目录号: P30-3031)
24. BV650抗小鼠/人CD11b( BioLegend ,目录号: 101239)
25. APC-R700大鼠抗小鼠CD45( BDHorizon TM ,目录号: 565478)
26. BV421大鼠抗小鼠F4 / 80(BD Biosciences;目录号: 565411)
27. PerCP-eFluor TM 710 抗小鼠 Ly6G(Invitrogen;目录号: 46-9668-82)
28. FITC抗小鼠CD3( BioLegend ,目录号: 100204)
29. FITC抗小鼠CD19( ImmunoTools ,目录号: 22220193S)
30. FITC抗小鼠CD49b( BioLegend ,目录号: 103503)
31. PE-Cyanine7抗小鼠iNOS (Invitrogen;目录号: 25-5920-80)
32. APC 抗小鼠 ARG1(Invitrogen,目录号: 17-3697-82)
33. BD Cytofix / Cytoperm TM (BD Biosciences,目录号: 51-2091K7)
34. BD PermWash TM (BD Biosciences,目录号: 51-2090K7)
35. 50 mL聚丙烯锥形管(Falcon,目录号:352070)
36. 氯胺酮( Livisto ,目录号:6680219)
37. 甲苯噻嗪( Animedica ,目录号:7630517)
38. Omnican F 注射器(Braun,目录号:91615025)
39. 一次性皮下注射针100 Sterican R(Braun,目录号:4657519)
40. Pen-Strep(Lonza,目录号:DE17-602E)
41. 红细胞裂解缓冲液(R&D,目录号:WL2000)
42. 吖啶橙/碘化丙啶染色剂( Biocat ,目录号:F23001 )
43. LB培养基(见食谱)
44. 含有 30% 甘油的 LB 培养基(参见食谱)


设备


1. 层流柜; EuroClone Safe Mate Eco 1.2 ( Politakis Laborgeräte ,目录号:EN 12 469)
2. 摇动培养箱(VWR,目录号:GFL 3031)
3. 贺利氏® HERAcell ® CO 2培养箱(Thermo Fisher Scientific,目录号: 3615-45 )
4. 光度计(Eppendorf, BioPhotometer D30,目录号:6133000001)
5. 离心机(Hettich Micro 200R 和Rotanta 460R,目录号:Z652113、Z623520)
6. CASY TT 计数系统(OMNI Life Science,目录号:TT-20A-2571)
7. CytoFLEX S V4-B4-R2-I2 流式细胞仪(13 个检测器,4 个激光器,Beckman Coulter,目录号:C01161)
8. LUNA 自动细胞计数仪 ( Biocat , L10001-LG)


软件


1. FlowJo v10.7.0(BD Biosciences, https: //www.flowjo.com/solutions/flowjo )
注意:任何用于分析流式细胞仪数据的软件包都可以与此协议一起使用。


程序


A. 鼠伤寒沙门氏菌 ( S. tm) 原液的制备
1. 取出一份表达红色荧光蛋白 (RFP) 的肠沙门氏菌鼠伤寒血清型 SL1344。
2. 在室温下解冻等分试样。
3. S. tm移液器放入 250 mL 锥形瓶中的 10 mL LB 培养基中。
4. 用锡箔盖住烧瓶的顶部。
5. 孵育过夜。
6. 第二天,将 50 μL 的隔夜培养物移液到 10 mL 的新鲜 LB 培养基中,放入250 mL Erlenmeyer 烧瓶中。
7. S.tm的隔夜文化。清洗和消毒250 毫升锥形瓶。
8. 用锡箔盖住烧瓶的顶部。
9. 在 200 rpm 和 37°C的摇动培养箱中孵育培养物1 – 2 小时。
10. 校准光度计,在一次性比色皿中使用 500 μL 的 LB 介质作为空白。 
11. 测量 OD 600 ,检查S. tm是否达到 0.5。
600在 0.5 – 0.7之间时, S. tm达到最佳对数生长期。
注意:如果 OD 600值低于 0.5,则如上所述继续在 250-mL 锥形瓶中孵育培养物,直到 OD 600值达到 0.5。值得注意的是,S.tm 密度每 20 分钟重复一次。如果 OD 600值高于 0.7,则用 LB 培养基 1:1 稀释培养物,然后在 250 mL 锥形瓶中孵育,直到 OD 600值为 0.5。
12. 将培养物转移到 50 mL 锥形管中。
13. S. tm培养物在室温下以4、967 × g离心5分钟。
14. 弃去上清液。
15. 将颗粒重新悬浮在 1 mL 新鲜制备的 LB 介质 + 30% 甘油中。
16. 在 0.5-mL Eppendorf 管中制备 50 µL 等分试样,并将其储存在 -20°C。


B. S. tm培养至最佳生长期
1. 解冻一份S. tm库存。
2. 将 10 µL 等分试样移液到250 mL 锥形瓶中的10 mL LB 培养基中,然后 用锡纸盖住烧瓶的顶部。
3. 在 200 rpm 和 37°C 的摇动培养箱中孵育过夜。
4. 第二天,将 50 µL 这种过夜培养物移液到250 mL 锥形瓶中的10 mL LB 培养基中,然后 用锡纸盖住烧瓶的顶部。
5. S.tm的隔夜文化。清洗和消毒250 mL 锥形瓶。
6. 在 200 rpm 和 37°C的摇动培养箱中孵育1 – 2 小时。
7. 校准光度计,在一次性比色皿中使用 500 μL 的 LB 介质作为空白。
8. 测量 OD 600以检查S. tm是否达到 0.5,这相当于最佳对数生长期。
注意:如果 OD 600值低于 0.5,则如上所述继续在 250-mL 锥形瓶中孵育培养物,直到 OD 600值达到 0.5。值得注意的是,S.tm 密度每 20 分钟重复一次。如果 OD 600值高于 0.7,则用 LB 培养基 1:1 稀释培养物,然后在 250 mL 锥形瓶中孵育,直到 OD 600值为 0.5。


C. 使用Casy计数系统计数可行的S. tm
1. 使用 45 µm 毛细管。
2. 装有 10 mL 新鲜Casy ton 缓冲液的新Casy杯来测量背景。
3. 选择背景测量程序(表 1 背景测量)。
4. 测量背景。这应该低于 30 个计数和 1 µm 的大小。否则,清洗系统。
5. 用 10 mL 的Casy ton缓冲液准备一个新的Casy杯,并添加 5 μL 的S. tm OD 600 0.5。
6. 轻轻摇晃。
7. 将样品放在测量单元下方。
8. 选择测量 1 – 3 µm 的程序(表 1 S. tm 测量)。
9. 措施。
10. 单击下一步,以获得可行计数/mL = 可行S. tm / mL 的数量。
600为 0.5的新鲜制备的 S.tm 培养物中的活菌计数应在 2.5 × 10 8 –3 × 10 8活菌计数/ mL 之间。
11. 测量完成后,取出样品杯,加入一个新的装有 10 mL Casy ton 缓冲液的Casy杯。
12. 执行五次Casy Clean。
13. 选择洗涤程序(表 1 洗涤程序)。
14. 洗涤完成后,再次检查背景。
15. 如果背景低于 30,可以关闭Casy计数系统。否则,继续洗涤。


表 1. CASY TT 计数系统程序
背景
测量 测量 
鼠伤寒沙门氏菌 洗涤程序
毛细管 45 µm X 轴:5 µm 45 µm X 轴:3 µm 45 µm X 轴:5 µm
样品量 200 µL 循环:1 200 µL 循环:3 200 µL 循环:10
稀释 1.001e+00 2.001e+03 1.001e+00
Y轴 汽车 汽车 汽车
评估光标 1.00 – 4.89 微米 0.75 – 2.93 微米 0.00 – 5.00 µm
规范。光标 0.5 – 4.89 微米 0.3 – 2.93 微米 0.00 – 5.00 µm
%计算 %通过碎片:开 %通过碎片:开 %通过碎片:开
聚合。正确的: 汽车 汽车 自动 P
界面 Par P.Feed : 开 Par P.Feed : 开 Par P.Feed : 开
打印模式 手动图形:开 手动图形:开 手动图形:开


D. 骨髓源性巨噬细胞 (BMDM) 的制备
Zanoni等人已经描述了 BMDM 的制备。 (2012;doi:10.21769/BioProtoc.225.)。


可以在以下位置找到演示该过程的视频: https://www.jove.com/de/v/52347/isolation-intravenous-injection-murine-bone-marrow-derived 。
图 1 显示了骨髓衍生巨噬细胞的产生和 S.tm 感染的示意图。


 
图 1. 实验装置的示意图,显示了骨髓衍生巨噬细胞的生成和S.tm感染。
未感染和感染的 BMDM 的不同形态在ScanR成像平台中可视化(图片由Demetz E. 友情提供)。


1. 腹腔注射 50 μL 的100 mg/kg 氯胺酮 + 10 mg/kg Xylazine来麻醉野生型 C57Bl/6N 小鼠。
注意:在小鼠腹腔注射中可以找到演示一般程序的视频: https ://researchanimaltraining.com/articles/intraperitoneal-injection-in-the-mouse/ 。
2. 对深度麻醉的小鼠进行安乐死。因此, 将一个大镊子放在麻醉小鼠头骨的底部后面, 并以 45° 角急剧向后拉尾巴。
3. 将动物固定在聚苯乙烯泡沫塑料板上,并用 75% 酒精喷洒动物表面。
4. 用无菌剪刀从脚后跟向上切割,从一条腿上去除皮肤和肌肉组织。
5. 在股骨头周围切开。
6. 在膝关节中间切开。小心不要伤到骨头。
7. 切开踝关节。
8. 用纸巾去除多余的肌肉。
9. 拉上大腿,将股骨头从髋关节中移除。
10. 将骨头放入冰上含有 1% 青霉素和 1% 链霉素的 PBS。
11. 移动到层流罩,并在冰上执行所有步骤。
12. 用一把无菌剪刀剪开骨头的末端。
13. 在 50 毫升猎鹰管上放置一个 40 微米的细胞过滤器。
14. 冲洗骨髓:
a. 使用一次性皮下注射针头和 5 毫升注射器 。
b. 用含有 1% 青霉素和 1% 链霉素的 PBS 填充注射器。
c. 将一根针放在打开的骨头的一端。
d. 将骨髓冲洗到 40 µm 细胞过滤器上。
e. 重复冲洗骨头,直到它完全变白。
15. 用含有 1% 青霉素和 1% 链霉素的 10 mL PBS 清洗细胞过滤器。
16. 使用注射器的柱塞,通过细胞过滤器拉紧细胞。
17. 用含有 1% 青霉素和 1% 链霉素的 10 mL PBS 清洗细胞过滤器。
18. 将装有冲洗过的骨髓的 50 mL 锥形管在 300 × g和 4°C 下离心 5 分钟。
19. 用双蒸水按 1:10 稀释红细胞裂解缓冲液
20. 弃去上清液。
21. 在 2 mL 稀释的红细胞裂解缓冲液中重新悬浮颗粒。
22. 在室温下孵育 3 分钟。
23. 在红细胞裂解缓冲液上加入 15 mL 的 PBS,其中含有 1% 青霉素和 1% 链霉素。
24. 将装有冲洗过的骨髓的 50 mL 锥形管在 300 × g和 4°C 下离心 5 分钟。
25. 弃去上清液。
26. 在含有 1% 青霉素和 1% 链霉素的 15 mL PBS 中重新悬浮颗粒。
27. 重复步骤 24-26。
28. 离心 50 mL 锥形管,弃去上清液。
29. 在 45 mL 的 DMEM 培养基中重新悬浮细胞颗粒,辅以 10% FBS、1% L-谷氨酰胺、1% 青霉素、1% 链霉素和50 ng/mL 重组鼠 M-CSF。
30. 将 15 mL 的细胞悬浮液移入三个 20 厘米的猎鹰培养皿中。
31. 每隔一天更换一次培养基。
32. 在第 5 天,可以收获细胞(程序 E)。


E. 细胞的收获和计数
1. 从细胞培养皿中取出培养基。
2. 用 10 mL 的 PBS 清洗细胞两次。
3. 添加 8 mL 的 DMEM 培养基,辅以 10% FBS 和 1% L-谷氨酰胺。
4. 使用一次性细胞刮刀刮擦细胞。
5. 用另外 2 mL 的 DMEM 培养基清洗盘子,辅以 10% FBS 和 1% L-谷氨酰胺。
6. 将细胞转移到 50 mL 锥形管中。
7. 关闭试管,将细胞倒置 2-3 次。
8. 将 9 μL 的细胞悬浮液放入 0.5 μL Eppendorf 管中。
9. 将 1 μL 吖啶橙/碘化丙啶染色溶液与细胞等分混合。
10. 将 10 μL 的混合物放入 Luna 细胞计数幻灯片中。
11. 使用 LUNA-FL 荧光和明场自动细胞计数器对细胞进行计数。
注意:在分离和培养两条后腿的骨髓后,从一只小鼠中获得大约 4.5 × 10 7 个细胞。
12. 将细胞播种在 6 厘米培养皿中,密度为 1.5 × 10 6 个细胞/mL,在 DMEM 培养基中添加 10% FBS 和 1% L-谷氨酰胺。
13. 为荧光减一 (FMO) 控制添加四个额外的培养皿(参见步骤 G3) 。
14. 让细胞在细胞培养箱中过夜。


F. S. tm体外感染骨髓来源的巨噬细胞
1. 在感染复数 10 (MOI 10) 处用S. tm感染 BMDM 。因此,添加比细胞多10倍的S. tm。
600为 0.5 的S.tm 培养物可用于制备新的 S.tm 等分试样(程序 A ),或丢弃。
2. 将细胞在细胞培养箱(5% CO 2 ,37°C)中孵育 1 小时。
3. 去除含有非吞噬S. tm的培养基。
4. 用 1 mL 的 PBS + 25 μg/mL 庆大霉素清洗细胞两次。
5. 添加 1 mL 的 DMEM 培养基,辅以 10% FBS、1% L-谷氨酰胺和 25 μg/mL 庆大霉素。
6. 在细胞培养箱中孵育细胞 4 小时。


G. 培养的 BMDM 的流式细胞术染色
1. 细胞外染色
a. 使用一次性细胞刮刀在培养基中刮取 BMDM。
b. × g和 4°C下离心5 分钟。
c. 弃去上清液。
d. 将颗粒重新悬浮在 1 mL 的 PBS 中,并将其转移到1.5 μL Eppendorf 管中。
e. 在 10,860 × g和 4°C 下短时间旋转 30 秒,使细胞沉淀。
f. 弃去上清液。
g. 将颗粒重新悬浮在 50 μL 的表面抗体混合物中(PBS 中的所有抗体 1:200;表 2)。


表 2. 流式细胞术抗体——细胞外染色。
抗体 克隆 公司 目录编号 表达于
CD3 FITC 17.A2 生物传奇 100204 T细胞
CD19 FITC PeCa1 免疫工具 22220193S B细胞
CD49b FITC HMa2 生物传奇 103503 NK细胞
CD11b BV650 M170 生物传奇 101239 中性粒细胞、单核细胞
CD45 APC-R700 30-F11 BD 地平线 565478 白细胞
F4/80 BV421 T45-2342 BD 地平线 565411 巨噬细胞
Ly6G PerCP-eFlour 710 1AB-Ly6g 英杰公司 46-9668-82 中性粒细胞


注意:为确保骨髓来源的巨噬细胞不受 T 细胞、B 细胞或 NK 细胞的污染,流式细胞仪面板中添加了针对 CD3、CD19 和 CD49b 的抗体。所有这些抗体都用 FITC 标记;因此,所有 FITC +细胞都可以排除在门控策略中(图 2)。 BMDM 培养物(未感染和感染 S.tm)的典型细胞分布如表 4 所示。


h. 4°C 避光孵育 10 分钟。
i. 用 500 μL 的 PBS 清洗。
j. 通过短时间旋转 (10,860 × g , 4°C, 30 s) 使细胞沉淀。
k. 弃去上清液。
l. 在 100 µL Cytofix / CytoPerm TM缓冲液中重悬沉淀,以渗透和固定细胞。
m. 4°C 避光孵育 20 分钟。
n. 用 Aqua bidest以 1:10稀释PermWash TM缓冲液。
o. 在 100 µL Cytofix / CytoPerm TM缓冲液上添加 500 µL 稀释的PermWash TM缓冲液。
p. 通过短时间旋转 (10,860 × g , 4°C, 30 s) 使细胞沉淀。
q. 弃去上清液。
2. 细胞内染色
a. PermWash TM缓冲液中制备细胞内抗体混合物( iNOS 1:100 和 ARG1 1:100;表 3)。


表 3. 流式细胞术抗体——细胞内染色。
抗体 克隆 公司 目录编号 表达于
iNOS PE-花青7 长鑫通 英杰公司 25-5920-80 促炎巨噬细胞
ARG1 装甲运兵车 A1exF5 英杰公司 17-3697-82 抗炎巨噬细胞


b. 将样品重新悬浮在 50 μL 的细胞内抗体混合物中。
c. 将样品在室温下避光孵育 45 分钟。
d. PermWash TM清洗细胞一次。通过短时间旋转 (10,860 × g , 4°C, 30 s) 使细胞沉淀。 
e. 丢弃上清液,将颗粒重新悬浮在 200 μL 的 PBS 中。
f. 通过 40 µm 过滤器将样品转移到平底 96 孔板中。
g. 直接在流式细胞仪中进行分析。


3. 荧光减一 (FMO) 对照
a. 如步骤 G1 中所述,在额外播种的未感染和感染的 BMDM 样本中执行 BMDM 的细胞外染色(参见步骤 E13)。
b. 在 100 µL Cytofix / CytoPerm TM缓冲液上用稀释的PermWash TM缓冲液洗涤后弃去上清液。
c. 在细胞内抗体混合物中重新悬浮一个未感染和一个感染的 BMDM 细胞颗粒,仅含有针对 ARG1 的抗体。重悬细胞内抗体混合物中的其他未感染和感染的 BMDM 细胞颗粒,仅使用针对iNOS的抗体。
d. 将样品在室温下避光孵育 45 分钟。
e. PermWash TM清洗细胞一次。 
f. 通过短时间旋转 (10860 × g , 4°C, 30 s) 使细胞沉淀。
g. 丢弃上清液,将颗粒重新悬浮在 200 μL 的 PBS 中。
h. 通过 40 µm 过滤器将样品转移到平底 96 孔板中。
i. 直接在流式细胞仪中进行分析。




 
图 2. 受感染的骨髓衍生巨噬细胞的门控策略 - 上图和未感染的骨髓衍生巨噬细胞 - 下图。 
在排除双峰后,白细胞被描述为 CD45 + 。排除 T 细胞、B 细胞和 NK 细胞(CD3 – CD19 – CD49b – ),巨噬细胞门控为 Ly6G – CD11b + F4/80 + 。根据研究问题,有可能将促炎巨噬细胞表征为表达iNOS的细胞,将抗炎巨噬细胞表征为表达 ARG1 的细胞。此外,含有 S.tm 的巨噬细胞的特点是在 PE 通道中表达红色荧光蛋白 (RFP)。


数据分析


FlowJo软件用于分析数据。门控策略如图 2 所示。
在由 C57BL/6N 小鼠产生的 BMDM 中,这些小鼠要么未感染,要么被 S.tm 感染,然后进一步孵育 4 小时,分析的细胞亚群的典型值如表 4 所示。


表 4. BMDM 培养的典型细胞分布。
细胞类型 门控 平均值± SEM 未感染 平均± SEM 感染
细胞 FSC-A 到 SSC-A 94.7 ± 2.1 95.2±2.1
单细胞 FSC-A 到 FSC-H 90.0 ± 2.7 91.7±2.2
白细胞 CD45 转 FSC-A 98.6 ± 1.3 99.5±0.3
FITC -阴性细胞 FITC(CD3、CD19、CD49b)转 FSC-A 98.6 ± 0.7 97.7±0.6
单核细胞 (Ly6G-) Ly6G 转 FSC-A 98.8 ± 0.7 98.4±0.5
巨噬细胞 F4/80 到 CD11b 98.0 ± 0.9 98.8±1.1
抗炎巨噬细胞 ARG1 到 FSC-A 1.8 ± 0.5 1.4±0.3
促炎巨噬细胞 iNOS转 FSC-A 4.2 ± 2.3 77.7±3.8
含有巨噬细胞的沙门氏菌 STR 转 FSC-A 0 24.7 ± 1.78


故障排除


A. S.tm的培养(见程序 A 和 B)
沙门氏菌 在 37°C 时生长最好,并且需要氧气才能实现最佳生长。因此,它们应该在覆盖有锡箔的锥形瓶中培养。必须严格保证37℃的温度。


B. BMDM 的制备(见程序D)
对于骨髓来源的巨噬细胞的产生,以无菌方式工作很重要。细胞只能在层流柜中处理。隔离整个胫骨和股骨很重要,并且仅在无菌条件下使用高压灭菌的剪刀和镊子将它们切开。


C. BMDM 的收获、计数和播种(参见程序 E)
通过刮擦收获 BMDM 需要相当温和,以获得高细胞活力。我们建议从身体上刮下来,不要对一次性细胞刮刀施加太大的力。
将细胞接种在无抗生素培养基中以进行感染。因此,在播种前一天,建议在放置在细胞培养箱中的细胞培养皿中孵育 5 mL 无抗生素培养基过夜,以确保培养基不受细菌污染。


D. BMDM 感染(见程序 F)
感染后,需要彻底冲洗掉未被吞噬的 S.tm。 _完全去除培养基很重要,并用PBS + 庆大霉素至少清洗细胞两次。可以使用显微镜来查看S. tm 是否仍然存在于培养物中。如果是,则应重复洗涤步骤。
重要的是在 PBS 中加入庆大霉素进行洗涤,并在 DMEM 中进一步孵育。庆大霉素通过阻断蛋白质生物合成来抑制 S.tm的增殖。


食谱


1. LB培养基
广告与
2% LB-肉汤
高压釜(121°C 20 分钟,50°C 10 分钟)
2. 含 30% 甘油的 LB 培养基
将 300 μL 的甘油添加到 700 μL 的 LB 培养基中


致谢


GW 得到了 Christian Doppler Society 的资助和 FWF(EPICROSS,I-3321)的 ERA-NET 资助,NB 得到了 FWF 博士学院项目 W1253 HOROS 的支持。
表达红色荧光蛋白的鼠伤寒沙门氏菌是由 Dirk Bumann教授(瑞士巴塞尔大学)赠送的礼物。该协议在 Fritsche等人之后进行了调整和修改。 (2008 年) 。


利益争夺


作者宣称没有利益冲突。


参考


1. Bhutta, ZA, Gaffey, MF, Crump, JA, Steele, D., Breiman, RF, Mintz, ED, Black, RE, Luby, SP 和 Levine, MM (2018)。伤寒:前进之路。 Am J Trop Med Hyg 99(3_Suppl):89-96。
2. 伯明翰, CL, Smith, AC, Bakowski, MA, Yoshimori, T. 和 Brumell, JH (2006)。自噬控制沙门氏菌感染以响应对含沙门氏菌的液泡的损害。 生物化学杂志 281(16):11374-11383 。
3. 波格丹,C.(2015 年)。先天性和适应性免疫中的一氧化氮合酶:更新。 趋势免疫36(3):161-178。
4. Brigo, N.、Pfeifhofer-Obermair, C.、Tymoszuk, P.、Demetz, E.、Engl, S.、Barros-Pinkelnig, M.、Dichtl, S.、Fischer, C.、Valente De Souza, L. , Petzer, V.等人。 (2021 年)。细胞因子介导的巨噬细胞中 ARG1 的调节及其对控制肠道沙门氏菌鼠伤寒血清型感染的影响。 单元格10(7)。
5. Buchmeier, NA 和 Heffron, F. (1991)。鼠伤寒沙门氏菌抑制巨噬细胞吞噬体-溶酶体融合。 感染免疫59(7):2232-2238。
6. De Muylder, G.、Daulouede, S.、Lecordier, L.、Uzureau, P.、Morias, Y.、Van Den Abbeele, J.、Caljon, G.、Herin, M.、Holzmuller, P.、Semballa, S.等人。 (2013)。布氏锥虫驱动蛋白重链通过触发宿主精氨酸酶活性促进寄生虫生长。 PLoS Pathog 9(10):e1003731。
7. El Kasmi, KC, Qualls, JE, Pesce, JT, Smith, AM, Thompson, RW, Henao-Tamayo, M., Basaraba, RJ, Konig, T., Schleicher, U., Koo, MS等。 (2008 年)。 Toll 样受体诱导的巨噬细胞精氨酸酶 1 阻碍了对细胞内病原体的有效免疫。 Nat Immunol 9(12):1399-1406。
8. Fritsche, G.、Nairz, M.、Werner, ER、Barton, HC 和 Weiss, G. (2008)。 Nramp1 功能通过抑制 IL-10 的形成来增加 iNOS 的表达。 Eur J Immunol 38(11):3060-3067。
9. Gordon, S. 和 Martinez-Pomares, L. (2017)。巨噬细胞的生理作用。 Pflugers Arch 469(3-4):365-374。
10. Hannemann, N., Cao, S., Eriksson, D., Schnelzer, A., Jordan, J., Eberhardt, M., Schleicher, U., Rech, J., Ramming, A., Uebe, S.,等。 (2019)。转录因子 Fra-1 靶向精氨酸酶 1 以增强巨噬细胞介导的关节炎炎症。 临床投资杂志 129(7):2669-2684。
11. Iniesta, V., Carcelen, J., Molano, I., Peixoto, PM, Redondo, E., Parra, P., Mangas, M., Monroy, I., Campo, ML, Nieto, CG,等。 (2005 年)。利什曼原虫主要感染期间的精氨酸酶 I 诱导介导疾病的发展。 感染免疫73(9):6085-6090 。
12. Lahiri, A.、Lahiri, A.、Iyer, N.、Das, P. 和 Chakravortty, D. (2010)。参观沙门氏菌感染的细胞生物学。 微生物感染12(11):809-818。
13. Mastroeni, P. 和 Grant, AJ (2011)。全身感染期间肠沙门氏菌在体内的传播:解开宿主和病原体的决定因素。 专家 Rev Mol Med 13:e12。
14. 米尔斯,CD(2012 年)。 M1 和 M2 巨噬细胞:健康和疾病的预言。 Crit Rev Immunol 32(6):463-488。
15. Mosser, DM 和 Edwards, JP (2008)。探索巨噬细胞活化的全谱。 Nat Rev Immunol 8(12):958-969。
16. 默里,PJ(2017)。巨噬细胞极化。 Annu Rev Physiol 79:541-566。
17. Nairz, M., Schleicher, U., Schroll, A., Sonnweber, T., Theurl, I., Ludwiczek, S., Talasz, H., Brandacher, G., Moser, PL, Muckenthaler, MU等. (2013)。一氧化氮介导的 ferroportin-1 调节控制沙门氏菌感染中的巨噬细胞铁稳态和免疫功能。 J Exp Med 210(5):855-873。
18. Navarre,WW,Zou,SB,Roy,H.,Xie,JL,Savchenko,A.,Singer,A.,Edvokimova,E.,Prost,LR,Kumar,R.,Ibba,M.,等。 (2010)。 PoxA、yjeK 和延伸因子 P 协同调节肠沙门氏菌的毒力和耐药性。 摩尔细胞39(2):209-221。
19. Ostuni, R. 和 Natoli, G. (2011)。巨噬细胞多样性和特化的转录控制。 Eur J Immunol 41(9):2486-2490。
20. Paduch, K.、Debus, A.、Rai, B.、Schleicher, U. 和 Bogdan, C. (2019)。在缺乏精氨酸酶 1 的情况下,皮肤利什曼病的解决和主要利什曼病的持续存在。 免疫杂志202(5):1453-1464。
21. Piccolo, V.、Curina, A.、Genua, M.、Ghisletti, S.、Simonatto, M.、Sabo, A.、Amati, B.、Ostuni, R. 和 Natoli, G.(2017 年)。相反的巨噬细胞极化程序显示出广泛的表观基因组和转录串扰。 Nat Immunol 18(5):530-540。
22. Schleicher, U., Paduch, K., Debus, A., Obermeyer, S., Konig, T., Kling, JC, Ribechini, E., Dudziak, D., Mougiakakos, D., Murray, PJ, et al . (2016 年)。 TNF 介导的骨髓细胞中精氨酸酶 1 表达的限制在感染部位触发 2 型 NO 合酶活性。 细胞代表15(5):1062-1075。
23. Weiss, G. 和 Schaible, UE (2015)。针对细胞内细菌的巨噬细胞防御机制。 免疫学第 264(1) 版:182-203。
24. Wu,A.,Tymoszuk,P.,Haschka,D.,Heeke,S.,Dichtl,S.,Petzer,V.,Seifert,M.,Hilbe,R.,Sopper,S.,Talasz,H.,等人。 (2017)。沙门氏菌利用锌通过调节 NF-kappaB 信号来破坏巨噬细胞的抗菌宿主防御。 感染免疫85(12)。
25. Zanoni, I.、Ostuni, R. 和 Granucci, F. (2012)。小鼠骨髓源性巨噬细胞 (BM-MFs) 的产生。 生物协议2(12):e225。


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引用:Brigo, N., Pfeifhofer-Obermair, C., Demetz, E., Tymoszuk, P. and Weiss, G. (2022). Flow Cytometric Characterization of Macrophages Infected in vitro with Salmonella enterica Serovar Typhimurium Expressing Red Fluorescent Protein. Bio-protocol 12(11): e4440. DOI: 10.21769/BioProtoc.4440.
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