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In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens
自身抗原和微生物源性抗原的体外抗原呈递测定   

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Abstract

Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and infected apoptotic cells.

Keywords: Antigen presentation(抗原呈递), Bone marrow derived dendritic cells(骨髓源性树突状细胞), CD4 T cells(CD4 T细胞), Self-antigens(自身抗原), Bacterial antigens(细菌抗原), Apoptotic cells(凋亡细胞)

Background

T cell lymphocytes express on their surface the T cell receptor (TCR), which allows the recognition of cellular (self) or microbial (non-self) antigens that are processed and presented as peptides bound to the major histocompatibility complex (MHC) molecules by antigen presenting cells (APC). APC are able to process antigens and to present them to T cells, and MHC-TCR interactions are critical steps for T cell activation during both infectious and autoimmune responses.

Previous works have described a mechanism of regulation of antigen presentation based on the stimulation of Pattern Recognition Receptors (PRRs), such as toll-like receptors (TLRs) (Blander and Medzhitov, 2004 and 2006). Indeed, TLR signals specifically from phagosomes containing microbial pathogens favor the presentation of non-self-antigens within MHC-II molecules. On the other hand, self-antigens generated after phagocytosis of apoptotic cells are directed to lysosomal degradation because of the absence of TLR stimuli. However, the segregation of self and non-self-antigens does not occur when both derive from infected apoptotic cells and are simultaneously carried by the same phagosome, which is optimally tailored by TLR signals for antigen presentation. Such mechanism of phagosome maturation and antigen presentation upon TLR triggering has been demonstrated in vitro using bone marrow derived dendritic cells (BMDC) and apoptotic murine B cells–either primary or A20 B cell line–previously incubated with the TLR4 ligand lipopolysaccharide (LPS), which is internalized by B cells and mimics bacterial infection (Blander and Medzhitov, 2004 and 2006; Campisi et al., 2016). Despite its elegance, this experimental system fails to reproduce bacterial invasion of the eukaryotic target cell. Furthermore, no T cell traceable antigens are present in the apoptotic cargo that internalized LPS.

I developed an in vitro alternative protocol where A20 cells are directly infected by the cell invasive bacteria Listeria monocytogenes expressing a recombinant antigen, allowing to assess the capacity of BMDC to present self and non-self-antigens derived from the same infected apoptotic cargo (Campisi et al., 2016).

Materials and Reagents

  1. Sterile pipette tips and serological pipettes (Fisher Scientific, FisherbrandTM)
  2. Optilux non-tissue culture10 cm Petri dishes (Corning, catalog number: 430591 )
  3. Sterile 50 and 15 ml conical tubes (Denville)
  4. 1 ml syringe with 26 G gauge needle (BD, catalog number: 309625 )
  5. 70 μm cell strainers (Fisher Scientific, FisherbrandTM, catalog number: 22-363-548 )
  6. Tissue culture 24 well plates (flat bottom) (Corning, Costar®, catalog number: 3524 )
  7. Tissue culture 96 well plates (flat bottom) (Corning, catalog number: 3595 )
  8. Sterile bacterial inoculating needles or loops
  9. Sterile 5 ml tubes with cap for bacterial culture (Corning, catalog number: 352058 )
  10. Tissue culture 6 well plates (flat bottom) (Corning, Costar®, catalog number: 3516 )
  11. 20 G gauge needle (BD, catalog number: 305175 )
  12. 3 ml syringe (BD, catalog number: 309656 )
  13. FACS tubes with rack (National Scientific, catalog number: TN0946-01R )
  14. Mice:
    1. Wild-type C57BL/6J mice
      Note: We initially purchased them from THE JACKSON LABORATORIES and then bred in the mouse facility of the Icahn School of Medicine at Mount Sinai for at least 5 years.
    2. OT-II TCR transgenic mice (strain B6.Cg-Tg(TcraTcrb)425Cbn/J) (THE JACKSON LABORATORIES, catalog number: 004194 ), which express the mouse alpha-chain and beta-chain T cell receptor that pairs with the CD4 coreceptor and is specific for an epitope derived from the chicken ovalbumin (OVA323-339) in the context of I-A b
    3. 1H3.1 TCR transgenic mice, which express the mouse alpha-chain and beta-chain T cell receptor that pairs with the CD4 coreceptor and is specific for the 52-68 fragment of the alpha-chain of I-E class II molecules (the Eα52-68 peptide) in the context of I-A b
  15. Antigen sources:
    1. Cell cargo: A20 cell line (ATCC, catalog number: TIB-208 )
    2. Bacteria: Listeria monocytogenes expressing ovalbumin (OVA) as a recombinant protein (Pope et al., 2001)
    3. Purified peptides: OVA329-337 (sequence ISQAVHAAHAEINEAGR) and Eα52-68 (sequence ASFEAQGALANIAVDKA)
  16. 70% ethanol
  17. 1x PBS (Sigma-Aldrich, catalog number: D8537 )
  18. Red blood cell lysis solution (Sigma-Aldrich, catalog number: R7757 )
  19. Bacterial growing medium: brain heart infusion (BHI) broth (BD, BactoTM, catalog number: 237500 )
  20. Ampicillin (Sigma-Aldrich, catalog number: A9393 )
  21. Anti-CD95 antibody, clone Jo2 (BD, BD Biosciences, catalog number: 554255 )
  22. Trypan blue stain (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  23. Fetal bovine serum (FBS)
  24. EDTA disodium dihydrate (Biological Industries, BI, catalog number: 41-922 ), to dissolve in PBS at pH = 8, stock solution 0.5 M
  25. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  26. Anti-CD4 magnetic microbeads for T cell positive selection (Miltenyi Biotec, catalog number: 130-049-201 )
  27. Carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher Scientific, eBioscienceTM, catalog number: 65-0850-84 )
  28. Anti-mouse CD4-APC, clone RM4-5 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 14-0042 )
  29. Optional: anti-mouse CD11c (clone N418), CD11b (clone M1/70) and MHC-II (clone M5/114.15.2) markers
  30. RPMI (Sigma-Aldrich, catalog number: R8758 )
  31. GM-CSF
    Note: We used to prepare GM-CSF using J558 cells transfected with GM-CSF cDNA (Liu et al., 2006, p.148), but recombinant GM-CSF can be also purchased.
  32. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  33. HEPES solution BioXtra, 1 M, pH 7.0-7.6 (Sigma-Aldrich, catalog number: H0887 )
  34. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 )
  35. MEM, nonessential amino acids (Sigma-Aldrich, catalog number: M7145 )
  36. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  37. IMDM (Sigma-Aldrich, catalog number: I3390 )
  38. Fc-block: rat anti-mouse CD16/32, clone 2.4G2 (BD, BD Biosciences, catalog number: 553141 )
  39. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: 13412 )
    Note: This product has been discontinued.
  40. BMDC medium (see Recipes)
  41. A20 cell medium (see Recipes)
  42. T cell medium (see Recipes)
  43. FACS buffer (see Recipes)

Equipment

  1. Single channel pipettes, 1, 20, 200 and 1,000 μl
  2. Scissors
  3. Forceps
  4. Laminar flow hood
  5. Bench top centrifuge
  6. Hemocytometer or automatic cell counter
  7. Cell incubator (37 °C, 5% CO2)
  8. Bacterial incubator (37 °C) with shaker
  9. Sterile 100 ml Erlenmeyer flasks
  10. Flasks for cell culture
  11. Optical density (OD) reader
  12. MACS columns, LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  13. MACS separators
    Note: I suggest QuadroMACS separator (Miltenyi Biotec, catalog number: 130-090-976 )
  14. Flow cytometer

Software

  1. Flow cytometer analysis software, FlowJo, LLC

Procedure

  1. Bone marrow isolation and dendritic cell differentiation and culture
    1. Sacrifice mice using CO2 accordingly to the institutional IACUC recommendations.
    2. Spray the entire mouse fur with 70% ethanol to avoid contaminations.
    3. Using a scissor cut the fur at the hip area and pull the skin down off the leg toward the foot.
    4. Once the skin removed, cut into pelvis and remove tibia and femur using a scissor.
    5. Hold tibia and femur with forceps and clean bones by cutting off all muscle/fat tissue using a scissor.
    Note: Steps A2 to A4 can be performed on a clean bench.
    1. Place the cleaned bones under a laminar flow hood -to work aseptically- into a 10 cm Petri dish with ice cold sterile PBS.
    2. Fill 1 ml syringe with fresh sterile cold PBS and, by holding the bone with the forceps, insert the needle into one hand and flush the bone marrow out the other hand in a second Petri dish containing 2-3 ml sterile cold PBS.
    3. Make a single cell suspension by passing through up and down with the same syringe.
    4. Refill the syringe with the PBS from the second Petri dish and repeat steps A6 and A7 until the bone becomes clear.
    5. Collect the cells in a conical 50 ml tube by filtering through a 70 μm cell strainer to remove any contamination of bone debris and tissue. Wash the Petri dish by pipetting up and down 1-2 ml PBS to be sure to recover all bone marrow cells, and add to the 50 ml tube through the 70 μm cell strainer.
    6. Adjust the final volume of the conical 50 ml tube to 30-40 ml with ice cold PBS.
    7. Pellet cells for 5 min at 435 x g.
    8. Remove the supernatant and resuspend in 1 ml of red blood cell lysis buffer, incubate for 1 min at room temperature, then add 25 ml of PBS to wash the cells and centrifuge for 5 min at 435 x g.
    9. Resuspend the pellet in 5 ml of Bone Marrow Dendritic Cells (BMDC) medium (see Recipes) and filter in a new 50 ml conical tube using a clean 70 μm cell strainer to remove dead cells. Count the cells using a hemocytometer or an automatic cell counter. Typically from 1 adult mouse 10 week old the yield should be 40 x 106 cells.
    10. Adjust the volume to a density of 106 cells/ml using BMDC medium, and plate 1 ml per well in a 24 well tissue culture plate.
    11. Incubate for 5 days in a cell incubator (37 °C, 5% CO2), adding 1 ml of BMDC medium at day 3 of culture. This is day 0. BMDC are semi-adherent, round cells that should not show extending dendrites when immature. BMDC purity can be assessed by flow cytometry using anti-mouse CD11c and MHC-II markers.
    12. Before use, count cells from 1 well after removing BMDC by pipetting up and down the medium very gently and washing out the well with 1 ml ice cold PBS. This cell number should be constant throughout the 24 wells. Centrifuge for 5 min at 279 x g.
    13. Add directly to the BMDC culture the phagocytic cargo (see Procedure B) resuspended in 200 μl of PBS or medium and then centrifuge the 24 well plate at 279 x g for 2 min.
      1. Alternatively, to get rid of contaminant cells that are not differentiated BMDC and adhere to the plate, BMDC can be removed from the 24 well plate as described in step A12 and replated in a new plate.
      2. Incubate BMDC (replated or not) with the phagocytic cargos for 8 h.
      3. Leave some BMDC without phagocytic cargo to be pulsed with peptide controls (see step A17 below).
    14. Harvest BMDC, collect in a conical tube and wash with cold PBS.
    15. Centrifuge for 5 min at 279 x g.
    16. Resuspend in T cell medium (see Recipes) at the concentration of 106 cells/ml.
    17. Plate in a 96 well plate 100 μl of BMDC immediately before adding the T cells. Pulsed unstimulated BMDC with 1 μg/ml of OVA329-337 and, in separate wells, Eα52-68 peptides. BMDC can be left on ice until T cells are ready.

  2. Phagocytic cargo preparation
    1. Listeria monocytogenes culture
      1. The night before the infection, prepare a bacteria pre-culture: aseptically pick a loopful of frozen bacteria from the stock using a needle or loop and inoculate into a 5 ml tube with 3 ml of BHI medium.
      2. Incubate for 16 h in a 37 °C incubator shaker at 250 rpm.
      3. The next day, dilute the overnight culture 1/20 in a sterile flask with 20 ml of fresh BHI medium. Cap and incubate in a 37 °C incubator shaker until the culture reaches the exponential phase (0.5 < OD600 < 1) (usually it takes 1 h). Determine the bacterial concentration (for Listeria monocytogenes, an OD600 = 1 corresponds to 5 x 108 bacteria/ml) and calculate the volume of culture needed to infect A20 cells and BMDC at a multiplicity of infection (moi) of 100.
      4. Transfer the appropriate volume of bacterial culture in one or more 50 ml tubes, centrifuge for 10 min at 3,113 x g, wash twice with sterile PBS.
      5. Add to cells 200 μl of bacteria in PBS or BMDC medium per well of cell culture.
        Note: Before addition to BMDC, bacteria should be killed by incubation for 2 h at 37 °C in PBS containing 50 μl of ampicillin, while A20 cells are infected with live bacteria.
    2. Preparation of A20 cells as apoptotic cell cargo
      1. A20 cells are grown in small flasks in A20 cell medium (see Recipes) in a cell incubator at 37 °C with 5% CO2.
      2. Three days before the assay, remove the A20 cells from the flask by pipetting up and down and transfer in a 50 ml conical tube.
      3. Count A20 cells, centrifuge at 279 x g for 5 min and resuspend them in fresh medium at 1 x 106 cells/ml.
      4. Plate 3 ml of cells per well in 6 well plates and place in the incubator.
      5. Induction of apoptosis in A20 cells:
        A20 cells will be rendered apoptotic by anti-CD95 (FasR) treatment, with or without previous bacteria infection, and then used as plain (= uninfected) or infected apoptotic cargos.
        1. Plain apoptotic cells:
          At day 5, remove and count A20 cells, centrifuge at 279 x g for 5 min, then resuspend and plate 3 ml in a 6 well plate at 1 x 106 cells/ml in fresh medium containing the anti-CD95 at the final concentration of 0.5 μg/ml.
          Incubate for 2 h in a cell incubator. The duration of anti-CD95 treatment can vary and should be precisely determined by each investigator. Apoptosis can be verified by flow cytometry after 7ADD and Annexin V staining (apoptotic cells should be Annexin V positive and 7ADD negative).
        2. Infected apoptotic cells
          At day 5, remove and count A20 cells, centrifuge at 279 x g for 5 min, then resuspend and plate 3 ml in a 6 well plate at 1 x 106 cells/ml in fresh medium. Add 200 μl of Listeria at a moi = 100. Incubate for 8 h in a cell incubator.
          After 8 h, add ampicillin at the final concentration of 50 μg/ml (to kill all the bacteria) and 0.5 μg/ml of anti-CD95 to the culture, cultivate for additional 2 h.
      6. Harvest and count apoptotic cells (they should not appear blue after trypan blue staining–more than 90% must be trypan blue negative–if they are, they cannot be used for the assay and the investigator should re-work the duration of the anti-CD95 treatment).
      7. Centrifuge at 279 x g for 5 min, and resuspend in 200 μl PBS or medium per well.
      8. Apoptotic cells are added immediately to the BMDC culture at a ratio of 1:2 (BMDC:apoptotic cells) for 8 h.

  3. CD4 T cell isolation and CFSE labeling
    1. Harvest spleens and prepare a cell suspension in a 10 cm Petri dish containing 5 ml cold PBS by pressing the tissue through a 70 μm cell strainer followed by homogenization using a 20 G needle and a 3 ml syringe. Make a single cell suspension by passing through up and down with the same syringe. Typically, one mouse spleen contains 1 x 108 splenocytes.
    2. Transfer the cell suspension in a 15 ml conical tube and centrifuge for 5 min at 435 x g.
    3. Optional: Remove the supernatant and resuspend in 1 ml of red blood lysis buffer, incubate for 1 min at room temperature, then add 13 ml of PBS to wash the cell and centrifuge for 5 min at 435 x g. This step can be skipped since red blood cells are eliminated during the step of CD4+ T cell positive selection.
    4. Incubate splenocytes in 1x PBS containing 1 μg/ml anti-Fc receptor, 2% FBS, 2 mM EDTA, 100 μg/ml penicillin, 100 μg/ml streptomycin and the anti-CD4 magnetic microbeads for CD4 T cell positive selection (Miltenyi Biotec), according to the manufacturer’s instructions.
    5. Proceed to CD4+ T cell positive selection by magnetic columns (Miltenyi Biotec), according to the manufacturer's instructions.
    6. Count and resuspend purified CD4 T cells in PBS at the concentration of 2 x 106 cells/ml.
    7. Add CFSE (final concentration 2.5 μM) very slowly to the cells while vortexing the tubes gently. Incubate for 10 min at 37 °C in the dark.
      Note: Leave some CD4 T cells unstained to perform flow cytometry compensations.
    8. Wash twice with PBS by completely filling the tube and centrifuging for 5 min at 435 x g. Successful CFSE staining of the cells should give a yellow/green pellet.
    9. Resuspend cells in T cell medium at the concentration of 2 x 106 cells/ml.
    10. Add 100 μl of CD4 T cells to the 96 well plates where BMDC were previously plated.
      Note: OT-II and 1H3.1 TCR transgenic CD4 T cells should be plated in separate wells to avoid antigen competition.
    11. Co-culture CD4 T cells and BMDC for 5 days.

  4. Assessing antigen presentation
    1. At day 5, wash twice the plate containing the co-culture of CD4 T cells and BMDC with 200 μl of FACS buffer (see Recipes) by centrifuging for 2 min at 778 x g. Discard the supernatant.
    2. Add 100 μl of anti-mouse CD4 (1/100) in FACS buffer and incubate for 20 min on ice in the dark.
    3. Wash twice with 200 μl of FACS buffer.
    4. Resuspend in 200 μl of FACS buffer and analyze by flow cytometry.

Data analysis

The following conditions should be prepared (at least in duplicate):

  1. OT-II CD4 T cells + BMDC that phagocytized plain apoptotic A20 cells = no T cell proliferation.
  2. OT-II CD4 T cells + BMDC that phagocytized Listeria-OVA infected apoptotic A20 cells = T cell proliferation.
  3. OT-II CD4 T cells + BMDC infected with Listeria-OVA = T cell proliferation.
  4. OT-II CD4 T cells + BMDC pulsed with OVA329-337 = positive control for OT-II CD4 T cell proliferation.
  5. OT-II CD4 T cells + BMDC pulsed with Eα52-68 peptide = negative control for OT-II CD4 T cell proliferation.
  6. 1H3.1 CD4 T cells + BMDC that phagocytized plain apoptotic A20 cells = no proliferation.
  7. 1H3.1 CD4 T cells + BMDC that phagocytized Listeria-OVA infected apoptotic A20 cells = T cell proliferation.
  8. 1H3.1 CD4 T cells + BMDC infected with Listeria-OVA = no T cell proliferation.
  9. 1H3.1 CD4 T cells + BMDC pulsed with OVA329-337 = negative control for 1H3.1 CD4 T cell proliferation.
  10. 1H3.1 CD4 T cells + BMDC pulsed with Eα52-68 peptide = positive control for 1H3.1 CD4 T cell proliferation.
  11. A20 cells are a Balb/c-derived B cell line that expresses I-E class II molecules. After phagocytosis of infected apoptotic A20 cells, the simultaneous compartmentalization of microbial and apoptotic cargo allows BMDC to process and present both the bacteria-derived antigen OVA329-337 and the cell-derived antigen Eα52-68, inducing the proliferation of OT-II and 1H3.1 CD4 T cells. On the other hand, in the absence of infection and TLR stimulus, antigens from plain apoptotic cells fail to be presented and no proliferation of 1H3.1 CD4 T cells should be observed (Figure 1).


    Figure 1. Regulation of antigen presentation by TLR activation. In this protocol, antigen presentation is assessed through proliferation of antigen-specific CD4 T cells diluting the CFSE after 5 days of incubation with antigen presenting cells (APC, here bone marrow derived dendritic cells), which phagocytized plain (A) or infected (C) apoptotic cells, or that have been infected with the bacteria Listeria monocytogenes expressing the recombinant protein ovalbumin (OVA) (B). I use as apoptotic cargos the A20 cell line, which expresses the Balb/c-derived Eα52-68 antigen recognized by TCR transgenic 1H3.1 CD4 T cells. OVA329-337 antigen derived from recombinant bacteria Listeria-monocytogenes is recognized by TCR transgenic OT-II CD4 T cells as microbial antigen.

  12. As shown in the Figure 1, phagocytosis of apoptotic cells is followed by lysosomal degradation of the cargo and no antigen presentation occurs (Figure 1A), while during bacterial infection TLR activation promotes phagosome maturation for optimal microbial antigen presentation (Figure 1B). When apoptotic cells are infected, both apoptotic and microbial cargos are found in the same phagosome where the presence of TLR signals favors antigen presentation of both self- and bacterial derived-antigens (Figure 1C).

Notes

  1. One mouse per strain (C57BL/6J, OT-II and 1H3.1 TCR transgenic) should give enough cells to perform the entire experiment.
  2. We observed down-regulation of the CD3 after antigen stimulation, thus an anti-CD3 staining is not necessary. For flow cytometry analysis, simply gate on live CD4+ T cells. This gating strategy is also enough to exclude BMDC. Alternatively, you can stain BMDC using anti-mouse CD11c and CD11b markers.
  3. Alternative versions of this protocol can be found in Blander and Medzhitov (2006) and Nair-Gupta et al. (2014). The latter paper describes in vitro antigen presentation assays in the context of CD8 T cells.

Recipes

  1. BMDC medium
    Complete RPMI with 10 ng/ml of GM-CSF and 5% FBS, plus 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 nM sodium pyruvate, 1x MEM nonessential amino acids, and 2.5 μM β-mercaptoethanol
  2. A20 cell medium
    Complete RPMI with 10% FBS, plus 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 nM sodium pyruvate, 1x MEM nonessential amino acids, and 2.5 μM β-mercaptoethanol
  3. T cell medium
    Complete IMDM with 10% FBS, plus 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 nM sodium pyruvate, 1x MEM nonessential amino acids, and 2.5 μM β-mercaptoethanol
  4. FACS buffer
    1x PBS with 2% FBS, plus 1 μg/ml Fc block and 0.1% NaN3

Acknowledgments

L.C. was supported by an Arthritis Foundation Research Fellowship Award. The protocol described herein was based on the paper Campisi et al. (2016). L.C. thanks Ivan Marazzi for advise and support. 1H3.1 TCR transgenic mice used in the paper Campisi et al. (2016) were a kind gift from Adrian Morelli to J. Blander’s laboratory under material transfer agreement (MTA).
The author declares no conflict of interest.

References

  1. Blander, J. M. and Medzhitov, R. (2004). Regulation of phagosome maturation by signals from toll-like receptors. Science 304(5673): 1014-1018.
  2. Blander, J. M. and Medzhitov, R. (2006). Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 440(7085): 808-812.
  3. Campisi, L., Barbet, G., Ding, Y., Esplugues, E., Flavell, R. A. and Blander, J. M. (2016). Apoptosis in response to microbial infection induces autoreactive TH17 cells. Nat Immunol 17(9): 1084-1092.
  4. Liu, K., Charalambous, A. and Steinman, R. M., (2006). Some biological features of dendritic cells in mouse. In: Fox, J., Barthold, S., Davisson, M., Newcomer, C., Quimby, F. and Smith, A. (Eds.). The Mouse in Biomedical Research. Academic Press, pp: 135-249.
  5. Nair-Gupta, P., Baccarini, A., Tung, N., Seyffer, F., Florey, O., Huang, Y., Banerjee, M., Overholtzer, M., Roche, P. A., Tampe, R., Brown, B. D., Amsen, D., Whiteheart, S. W. and Blander, J. M. (2014). TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation. Cell 158(3): 506-521.
  6. Pope, C., Kim, S. K., Marzo, A., Masopust, D., Williams, K., Jiang, J., Shen, H. and Lefrançois, L. (2001). Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J Immunol 166(5): 3402-9.

简介

抗原呈递细胞(APC)能够处理和呈递来自不同来源的T细胞抗原。这种机制是高度调节的,特别是通过Patter Recognition Receptor(PRR)信号。在这里,我详细说明了一种设计用于评估体外的APC方案,用于展示来源于细菌,凋亡和感染的凋亡细胞的抗原。

背景 T细胞淋巴细胞在其表面上表达T细胞受体(TCR),其允许识别作为与主要组织相容性复合物(MHC)分子结合的抗原加工和呈递的抗原的细胞(自身)或微生物(非自身)抗原)呈递细胞(APC)。 APC能够处理抗原并将其呈递给T细胞,并且MHC-TCR相互作用是感染和自身免疫应答期间T细胞活化的关键步骤。
&NBSP;以前的作品已经描述了基于刺激模式识别受体(PRR),例如toll样受体(TLR)(Blander和Medzhitov,2004和2006)的抗原呈递的调节机制。实际上,特异性地来自含有微生物病原体的吞噬体的TLR信号有利于在MHC-II分子内呈递非自身抗原。另一方面,凋亡细胞吞噬后产生的自身抗原由于不存在TLR刺激而导致溶酶体降解。然而,当两者都来自感染的凋亡细胞并且同时由相同的吞噬体携带时,自身和非自身抗原的分离不会发生,其由针对抗原呈递的TLR信号最佳地定制。已经使用骨髓来源的树突状细胞(BMDC)和凋亡性小鼠B细胞 - 原代或A20B细胞系 - 预先与培养基进行体外实验,证明了TLR触发后的吞噬体成熟和抗原呈递的这种机理体外 TLR4配体脂多糖(LPS),其被B细胞内化并模拟细菌感染(Blander和Medzhitov,2004和2006; Campisi等人,2016)。尽管它的优雅,这个实验系统不能再现真核细胞的细菌侵袭。此外,内含LPS的凋亡货物中不存在T细胞可追踪抗原。
&NBSP;我开发了体外替代方案,其中A20细胞被表达重组抗原的细胞侵袭性细菌单核细胞增生利斯特氏菌直接感染,允许评估BMDC呈现的能力来自相同感染的凋亡物质的自身和非自身抗原(Campisi等人,2016)。

关键字:抗原呈递, 骨髓源性树突状细胞, CD4 T细胞, 自身抗原, 细菌抗原, 凋亡细胞

材料和试剂

  1. 无菌移液器吸头和血清移液器(Fisher Scientific,Fisherbrand TM
  2. Optilux非组织培养10厘米培养皿(康宁,目录号:430591)
  3. 无菌50和15毫升锥形管(Denville)
  4. 1 ml注射器,带26 G标针(BD,目录号:309625)
  5. 70微米的细胞过滤器(Fisher Scientific,Fisherbrand TM,目录号:22-363-548)
  6. 组织培养24孔板(平底)(Corning,Costar ®,目录号:3524)
  7. 组织培养96孔板(平底)(康宁,目录号:3595)
  8. 无菌细菌接种针或环
  9. 无菌5毫升带细菌培养瓶的管(康宁,目录号:352058)
  10. 组织培养6孔板(平底)(Corning,Costar ®,目录号:3516)
  11. 20 G规针(BD,目录号:305175)
  12. 3 ml注射器(BD,目录号:309656)
  13. 带机架的FACS管(国家科学,目录号:TN0946-01R)
  14. 老鼠:
    1. 野生型C57BL / 6J小鼠
      注意:我们最初从“杰克逊实验室”购买了这些产品,然后在西奈山的伊坎医学院的鼠标设备中培育至少5年。
    2. 表达与CD4配对的小鼠α链和β链T细胞受体的OT-IITCR转基因小鼠(菌株B6.Cg-Tg(TcraTcrb)425Cbn / J)(THE JACKSON LABORATORIES,目录号:004194)并且在IAb的情况下对来自鸡卵白蛋白(OVA 323-339 )的表位是特异性的
    3. 1H3.1 TCR转基因小鼠,其表达与CD4受体配对的小鼠α链和β链T细胞受体,并且对IE II类分子的α链的52-68片段是特异性的(Eα<亚> 52-68 肽)
  15. 抗原来源:
    1. 细胞载体:A20细胞系(ATCC,目录号:TIB-208)
    2. 细菌:表达卵白蛋白(OVA)作为重组蛋白的单核细胞增生利斯特氏菌(PEEE等人,2001)
    3. 纯化肽:OVA 329-337(序列ISQAVHAAHAEINEAGR)和Eα52-68(序列ASFEAQGALANIAVDKA)
  16. 70%乙醇
  17. 1x PBS(Sigma-Aldrich,目录号:D8537)
  18. 红细胞裂解液(Sigma-Aldrich,目录号:R7757)
  19. 细菌生长培养基:脑心脏输注(BHI)肉汤(BD,Bacto TM,目录号:237500)
  20. 氨苄青霉素(Sigma-Aldrich,目录号:A9393)
  21. 抗CD95抗体,克隆Jo2(BD,BD Biosciences,目录号:554255)
  22. 台盼蓝染色(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
  23. 胎牛血清(FBS)
  24. EDTA二钠二水合物(Biological Industries,BI,目录号:41-922),溶于pH = 8的PBS中,储备溶液0.5M
  25. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  26. 用于T细胞阳性选择的抗CD4磁性微珠(Miltenyi Biotec,目录号:130-049-201)
  27. 羧基荧光素琥珀酰亚胺酯(CFSE)(Thermo Fisher Scientific,eBioscience TM,目录号:65-0850-84)
  28. 抗小鼠CD4-APC,克隆RM4-5(Thermo Fisher Scientific,eBioscience TM,目录号:14-0042)
  29. 可选:抗小鼠CD11c(克隆N418),CD11b(克隆M1 / 70)和MHC-II(克隆M5 / 114.15.2)标记物
  30. RPMI(Sigma-Aldrich,目录号:R8758)
  31. GM-CSF
    注意:我们曾用GM-CSF cDNA转染的J558细胞制备GM-CSF(Liu等,2006,p.148),但也可购买重组GM-CSF。 />
  32. L-谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  33. HEPES溶液bioxtra,1M,pH 7.0-7.6(Sigma-Aldrich,目录号:H0887)
  34. 丙酮酸钠(Sigma-Aldrich,目录号:S8636)
  35. MEM,非必需氨基酸(Sigma-Aldrich,目录号:M7145)
  36. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  37. IMDM(Sigma-Aldrich,目录号:I3390)
  38. Fc区:大鼠抗小鼠CD16 / 32,克隆2.4G2(BD,BD Biosciences,目录号:553141)
  39. 叠氮化钠(NaN 3 N 3)(Sigma-Aldrich,目录号:13412))
    注意:本产品已停产。
  40. BMDC培养基(见食谱)
  41. A20细胞培养基(参见食谱)
  42. T细胞培养基(参见食谱)
  43. FACS缓冲区(见配方)

设备

  1. 单通道移液器,1,20,200和1,000μl
  2. 剪刀
  3. 镊子
  4. 层流罩
  5. 台式离心机
  6. 血细胞计数器或自动细胞计数器
  7. 细胞培养箱(37℃,5%CO 2
  8. 细菌培养箱(37°C),带摇床
  9. 无菌100ml锥形瓶
  10. 烧瓶用于细胞培养
  11. 光密度(OD)读者
  12. MACS列,LS列(Miltenyi Biotec,目录号:130-042-401)
  13. MACS分隔符
    注意:我建议使用QuadroMACS分离器(Miltenyi Biotec,目录号:130-090-976)
  14. 流式细胞仪

软件

  1. 流式细胞仪分析软件,FlowJo,LLC

程序

  1. 骨髓分离和树突细胞分化培养
    1. 根据机构IACUC建议,使用CO 2牺牲小鼠。
    2. 用70%乙醇喷洒整个小鼠毛皮以避免污染。
    3. 使用剪刀在臀部区域切割毛皮,将皮肤从腿部向脚部向下拉。
    4. 一旦皮肤被去除,切成骨盆,并用剪刀去除胫骨和股骨。
    5. 使用剪刀切断所有肌肉/脂肪组织,用镊子和干净的骨骼夹持胫骨和股骨。
    注意:步骤A2至A4可以在干净的工作台上进行。
    1. 将清洁的骨头放置在层流罩下方,无菌无菌地加入到带有冰冷无菌PBS的10厘米培养皿中。
    2. 用新鲜无菌冷PBS填充1ml注射器,通过用镊子夹住骨头,将针插入一只手,另一只手将另一只手冲入含有2-3ml无菌冷PBS的第二培养皿中。
    3. 使用相同的注射器通过上下传递制作单细胞悬浮液。
    4. 用PBS从第二培养皿补充注射器,重复步骤A6和A7,直到骨骼变得清晰。
    5. 通过过滤70μm细胞过滤器将细胞收集在圆锥形的50ml管中,以去除骨碎片和组织的任何污染。通过上下移动1-2ml PBS洗涤培养皿,以确保回收所有骨髓细胞,并通过70μm细胞过滤器加入50ml管中。
    6. 使用冰冷的PBS将锥形50ml管的最终体积调节至30-40ml。
    7. 颗粒细胞在435 x g下5分钟。
    8. 去除上清液并重悬于1ml红细胞裂解缓冲液中,室温孵育1分钟,然后加入25ml PBS洗涤细胞,并以435g离心5分钟。
    9. 将沉淀重悬于5ml骨髓树突状细胞(BMDC)培养基(参见食谱)中,并使用干净的70μm细胞过滤器在新的50ml锥形管中过滤以除去死细胞。使用血细胞计数器或自动细胞计数器计数细胞。通常来自1只成年小鼠10周龄,产量应为40×10 6个细胞。
    10. 使用BMDC培养基将体积调节至10×10 6细胞/ ml的密度,并在24孔组织培养板中每孔1ml板。
    11. 在细胞培养箱(37℃,5%CO 2)中孵育5天,在培养的第3天加入1ml BMDC培养基。这是第0天。BMDC是半粘附的圆形细胞,当不成熟时不应显示延伸的树突。 BMDC纯度可以通过使用抗小鼠CD11c和MHC-II标记的流式细胞术进行评估
    12. 在使用前,通过轻轻移液培养基去除BMDC后,从1孔计数细胞,并用1ml冰冷的PBS洗涤孔。这个细胞数量应该在整个24个孔中是恒定的。在279 xg离心5分钟。
    13. 将直接添加到BMDC培养物中的吞噬货物(参见方法B)重悬于200μlPBS或培养基中,然后以279×g离心24孔板2分钟。
      1. 或者,为了除去未分化的BMDC并附着于板的污染细胞,可以如步骤A12所述从24孔板除去BMDC,并在新板中重新填充。
      2. 孵育BMDC(补充或不补充)与吞噬货物8小时。
      3. 留下一些没有吞噬量的BMDC用肽控制剂脉冲(见下面的步骤A17)。
    14. 收获BMDC,收集在锥形管中,用冷PBS洗涤
    15. 离心5分钟,279
    16. 以10 6细胞/ ml的浓度重悬于T细胞培养基(参见食谱)。
    17. 在加入T细胞之前,在96孔板中加入100μlBMDC。脉冲未刺激的BMDC,其具有1μg/ ml的OVA 329-337和在分开的孔中的Eα52-68肽。 BMDC可以留在冰上,直到T细胞准备就绪
  2. 吞噬货物准备
    1. 单核细胞增生利斯特氏菌文化
      1. 感染前一天,准备细菌预培养:用针或环无菌挑取一批来自原种的冷冻细菌,并用3ml BHI培养基接种到5ml的管中。
      2. 在37℃的培养箱中以250rpm的速度孵育16小时
      3. 第二天,用无菌烧瓶稀释20ml新鲜BHI培养基的过夜培养物1/20。盖上并在37℃培养箱振荡器中孵育,直到培养物达到指数期(0.5 1对应于5×10 8细菌/ ml),并计算细菌浓度需要感染A20细胞和BMDC多种感染(moi)为100的文化
      4. 将适量体积的细菌培养物转移到一个或多个50ml管中,以3,113×g离心10分钟,用无菌PBS洗涤两次。
      5. 在细胞培养物中每孔加入PBS或BMDC培养基中的细胞200μl。
        注意:在加入BMDC之前,细菌应该在37℃下在含有50μl氨苄青霉素的PBS中孵育2小时而灭活,而A20细胞用活细菌感染。
    2. A20细胞作为细胞凋亡细胞的制备
      1. A20细胞在37℃,5%CO 2的细胞培养箱中,在A20细胞培养基(参见食谱)的小烧瓶中生长。
      2. 在测定前三天,通过上下移液移出瓶中的A20细胞,并在50ml锥形管中转移。
      3. 计数A20细胞,以279 x g离心5分钟,并将其重悬于1×10 6细胞/ ml的新鲜培养基中。
      4. 在6孔板中每孔3ml细胞板,并置于培养箱中
      5. 诱导A20细胞凋亡:
        A20细胞通过抗CD95(FasR)处理而被凋亡,有或没有以前的细菌感染,然后用作平原(=未感染)或感染的凋亡载体。
        1. 细胞凋亡细胞:
          在第5天,取出并计数A20细胞,以279×g离心5分钟,然后将其重新悬浮并以1×10 6细胞/孔加入6孔板中3ml, ml,含最终浓度为0.5μg/ ml的抗CD95的新鲜培养基 在细胞培养箱中孵育2小时。抗CD95治疗的持续时间可以变化,应由每位研究者精确确定。细胞凋亡可以通过7ADD和Annexin V染色后的流式细胞术检测(凋亡细胞应为Annexin V阳性和7ADD阴性)。
        2. 感染的凋亡细胞
          在第5天,取出并计数A20细胞,以279×g离心5分钟,然后将其重新悬浮并以1×10 6细胞/孔加入6孔板中3ml, ml的新鲜培养基。在moi = 100中加入200μl的李斯特菌。在细胞培养箱中孵育8小时。
          8小时后,加入终浓度为50μg/ ml的氨苄青霉素(杀死所有细菌)和0.5μg/ ml抗CD95至培养物中,再培养2小时。
      6. 收获和计数凋亡细胞(台盼蓝染色后不应该出现蓝色 - 超过90%必须是台盼蓝,否则不能用于测定,研究人员应重新进行抗紫外线的持续时间, CD95治疗)
      7. 以279 x g离心5分钟,并重悬于200μlPBS或培养基中。
      8. 凋亡细胞以1:2(BMDC:凋亡细胞)的比例立即加入BMDC培养物中8小时。

  3. CD4 T细胞分离和CFSE标记
    1. 收获脾,并在含有5ml冷PBS的10cm陪替氏培养皿中制备细胞悬浮液,通过将组织挤压通过70μm细胞过滤器,然后使用20G针头和3ml注射器均质化。通过使用相同的注射器上下通过单细胞悬浮液。通常,一只小鼠脾脏含有1×10 8个脾细胞。
    2. 将细胞悬浮液转移到15ml锥形管中,并以435g离心5分钟。
    3. 可选:取上清液并重悬于1 ml红血裂解缓冲液中,室温孵育1 min,然后加入13 ml PBS洗涤细胞,并以435 x g离心5 min。可以跳过这一步,因为在CD4 + sup + T细胞阳性选择的步骤中消除红细胞。
    4. 在含有1μg/ ml抗Fc受体,2%FBS,2mM EDTA,100μg/ ml青霉素,100μg/ ml链霉素和用于CD4T细胞阳性选择的抗CD4磁性微珠的1ml PBS中孵育脾细胞(Miltenyi Biotec ),根据制造商的说明。
    5. 根据制造商的说明,按磁性柱(Miltenyi Biotec)进行CD4 + T细胞阳性选择。
    6. 以2×10 6细胞/ ml的浓度计数并重新悬浮于PBS中纯化的CD4T细胞。
    7. 将CFSE(终浓度为2.5μM)缓慢加入细胞,同时轻轻涡旋管。在黑暗中37℃孵育10分钟。
      注意:使一些CD4 T细胞未染色,以进行流式细胞术补偿。
    8. 通过完全填充管并用435×g离心5分钟,用PBS洗涤两次。细胞成功的CFSE染色应该会产生黄色/绿色颗粒
    9. 以2×10 6细胞/ ml的浓度将细胞重悬于T细胞培养基中。
    10. 将100μlCD4 T细胞添加到先前接种BMDC的96孔板中。
      注意:OT-II和1H3.1 TCR转基因CD4T细胞应铺在分开的孔中以避免抗原竞争。
    11. 共培养CD4T细胞和BMDC 5天
  4. 评估抗原呈递
    1. 在第5天,用含有200μlFACS缓冲液(参见食谱)的CD4T细胞和BMDC的共培养物的板洗涤两次,通过在778×g离心2分钟。丢弃上清液。
    2. 在FACS缓冲液中加入100μl抗小鼠CD4(1/100),并在黑暗中在冰上孵育20分钟。
    3. 用200μlFACS缓冲液洗涤两次。
    4. 重悬于200μlFACS缓冲液中,流式细胞术检测

数据分析

应准备以下条件(至少重复):

  1. OT-II CD4 T细胞+吞噬细胞凋亡A20细胞的BMDC =无T细胞增殖。
  2. 吞噬吞噬李斯特菌的OT-II CD4 T细胞+ BMDC -OVA感染的凋亡A20细胞= T细胞增殖。
  3. OT-II CD4 T细胞+利用李斯特菌感染的BMDC -OVA = T细胞增殖。
  4. OT-II CD4 T细胞+用OVA 329-337脉冲的BMDC = OT-II CD4 T细胞增殖阳性对照。
  5. 用Eα52-68肽脉冲的OT-II CD4 T细胞+ BMDC = OT-II CD4 T细胞增殖的阴性对照。
  6. 1H3.1吞噬细胞凋亡A20细胞的CD4 T细胞+ BMDC =无增殖
  7. 1H3.1吞噬李斯特菌的CD4T细胞+ BMDC -OVA感染的凋亡A20细胞= T细胞增殖。
  8. 1H3.1用李斯特氏菌感染的CD4T细胞+ BMDC -OVA =无T细胞增殖。
  9. 1H3.1用OVA 329-337脉冲的CD4T细胞+ BMDC = 1H3.1 CD4 T细胞增殖的阴性对照。
  10. 1H3.1用Eα52-68肽脉冲的CD4T细胞+ BMDC = 1H3.1 CD4 T细胞增殖的阳性对照。
  11. A20细胞是表达I-E II类分子的Balb / c衍生的B细胞系。在感染的凋亡A20细胞吞噬后,微生物和凋亡物质的同时分隔使BMDC能够处理并呈递细菌衍生的抗原OVA 329-337和细胞衍生的抗原Eα52 -68 ,诱导OT-II和1H3.1 CD4T细胞的增殖。另一方面,在没有感染和TLR刺激的情况下,来自细胞凋亡细胞的抗原不能呈现,并且不应该观察到1H3.1 CD4T细胞的增殖(图1)。

    图1.通过TLR活化的抗原呈递的调节在该方案中,通过抗原特异性CD4T细胞的增殖来评估抗原呈递,所述抗原特异性CD4T细胞在与抗原呈递细胞孵育5天之后稀释CFSE(APC, (A)或感染(C)凋亡细胞,或已被表达重组蛋白卵白蛋白(OVA)(B)的细菌单核细胞增生利斯特氏杆菌感染的骨髓衍生树突状细胞) 。我使用A20细胞系作为凋亡载体,其表达由TCR转基因1H3.1 CD4T细胞识别的Balb / c衍生的Eα52-68抗原。来自重组细菌李斯特菌 - 单核细胞增生李斯霉素的OVA 329-337抗原被TCR转基因OT-II CD4 T细胞识别为微生物抗原。

  12. 如图1所示,凋亡细胞的吞噬作用随后是货物的溶酶体降解,并且不发生抗原呈递(图1A),而在细菌感染期间TLR活化促进吞噬体成熟以获得最佳微生物抗原呈递(图1B)。当凋亡细胞被感染时,在相同的吞噬体中发现凋亡细胞和微生物的货物,其中TLR信号的存在有利于自身和细菌衍生抗原的抗原呈递(图1C)。

笔记

  1. 每个菌株一个小鼠(C57BL / 6J,OT-II和1H3.1 TCR转基因)应该给足够的细胞进行整个实验。
  2. 我们观察到抗原刺激后CD3的下调,因此不需要进行抗CD3染色。对于流式细胞术分析,只需在活CD4 + sup + T细胞上敲门。这种门控策略也足以排除BMDC。或者,您可以使用抗小鼠CD11c和CD11b标记染色BMDC。
  3. 该协议的替代版本可以在Blander和Medzhitov(2006)和Nair-Gupta等人中找到。 (2014)。后一篇论文描述了在CD8 T细胞的上下文中的体外抗原呈递测定。

食谱

  1. BMDC媒体
    用10ng / ml GM-CSF和5%FBS,加100μg/ ml青霉素,100μg/ ml链霉素,2mM L-谷氨酰胺,10mM HEPES,1nM丙酮酸钠,1×MEM非必需氨基酸,和2.5μMβ-巯基乙醇
  2. A20细胞培养基 使用10%FBS,加100μg/ ml青霉素,100μg/ ml链霉素,2mM L-谷氨酰胺,10mM HEPES,1nM丙酮酸钠,1×MEM非必需氨基酸和2.5μMβ-巯基乙醇的RPMI, >
  3. T细胞培养基 用10%FBS,100μg/ ml青霉素,100μg/ ml链霉素,2mM L-谷氨酰胺,10mM HEPES,1nM丙酮酸钠,1×MEM非必需氨基酸和2.5μMβ-巯基乙醇完成IMDM, >
  4. FACS缓冲区
    具有2%FBS的1x PBS,加上1μg/ ml Fc阻滞物和0.1%NaN 3

致谢

L.C.得到了关节炎基金会研究奖学金的支持。本文描述的协议是基于Campisi等人的论文。 (2016)。 L.C.感谢Ivan Marazzi的建议和支持。 1H3.1用于Campisi等人的TCR转基因小鼠。 (2016)是从Adrian Morelli到J. Blander实验室的物质转让协议(MTA)的礼物。
作者声明没有利益冲突。

参考

  1. Blander,JM和Medzhitov,R。(2004)。&nbsp; 通过来自收费样受体的信号调节吞噬体成熟。科学 304(5673):1014-1018。
  2. Blander,JM和Medzhitov,R。(2006)。&nbsp; Toll-dependent selection of microbial antigens for presentation by dendritic cells。自然 440(7085):808-812。
  3. Campisi,L.,Barbet,G.,Ding,Y.,Esplugues,E.,Flavell,RA and Blander,JM(2016)。&lt; a class =“ke-insertfile”href =“http:// www .ncbi.nlm.nih.gov / pubmed / 27455420“target =”_ blank“>响应于微生物感染的细胞凋亡诱导自身反应性TH17细胞。 Nat Immunol 17(9):1084- 1092.
  4. Liu,K.,Charalambous,A.and Steinman,RM,(2006)。&nbsp; 小鼠中树突状细胞的一些生物学特征。在Fox:J.,Barthold,S.,Davisson,M 。,Newcomer,C.,Quimby,F.和Smith,A.(Eds。)。生物医学研究中的鼠标。 Academic Press ,pp:135-249。
  5. Nair-Gupta,P.,Baccarini,A.,Tung,N.,Seyffer,F.,Florey,O.,Huang,Y.,Banerjee,M.,Overholtzer,M.,Roche,PA,Tampe, ,Brown,BD,Amsen,D.,Whiteheart,SW and Blander,JM(2014)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed / 25083866“target =”_ blank“> TLR信号从内体回收室诱导吞噬细胞MHC-I递送以允许交叉表达。细胞 158(3):506-521。 br />
  6. 教授,C.,Kim,SK,Marzo,A.,Masopust,D.,Williams,K.,Jiang,J.,Shen,H。和Lefrançois,L。(2001)。&nbsp; CD8 T细胞对单核细胞增生利斯特氏菌感染反应的器官特异性调节。 em Immuno 166(5):3402-9。
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引用:Campisi, L. (2017). In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens. Bio-protocol 7(11): e2307. DOI: 10.21769/BioProtoc.2307.
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