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Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs)

人单核细胞衍生的树突状细胞(MDDC)捕获HIV-1和葡聚糖的定量测定

Mickaël M. Ménager Mickaël M. Ménager
DL Dan R. Littman

发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2004 浏览次数: 10979

评审: Kristopher MarjonVaibhav B ShahValeria Lulla

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Abstract

The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.

Background

This protocol was developed in order to assess the changes of HIV-1 internalization upon disruption of actin nucleation in human monocyte derived dendritic cells. Following a shRNA screen to identify genes important for HIV-1 transfer from dendritic cells to T cells, we observed that a disruption of actin nucleation leads to a switch from actin rich dendrites to blebs, due to an excess of actomyosin contraction. As a consequence, a decrease of HIV-1 transfer and an increase of HIV-1 internalization due to bleb retraction-driven macropinocytosis were observed. We concluded that effectors of actin nucleation and stabilization were key to maintain HIV-1 on actin-rich dendrites and to limit its endocytosis, for efficient transfer to T lymphocytes (Menager and Littman, 2016).

Materials and Reagents

  1. Production of HIV-1 particles
    1. 10 cm plate (100 x 20 mm) (Corning, Falcon®, catalog number: 353003 )
    2. 15 cm plate (150 x 20 mm) (Corning, Falcon®, catalog number: 353025 )
    3. 15 ml conical tube
    4. 33 mm Millex-HV syringe filter, PVDF, 0.45 μm (EMD Millipore, catalog number: SLHV033RS )
    5. 60 ml syringe with BD Luer-LokTM tip (BD, Luer-LokTM, catalog number: 309653 )
    6. 50 ml high clarity PP centrifuge tube, conical bottom, sterile, 25/bag, 500/case (Corning, Falcon®, catalog number: 352070 )
    7. 293FT cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: R700-07 )
    8. X4-HIV-Gag-iGFP plasmid
      Note: Derived from HIV-1 molecular clone NL4-3 modified to insert the GFP protein in between the MA and CA domains of Gag as previously described (Hubner et al., 2007).
    9. Dulbecco’s modification of Eagles medium (DMEM) (Mediatech, catalog number: 10-017-CV )
    10. Fetal bovine serum (FBS)
    11. MEM amino acid solution (GE Healthcare, HyCloneTM, catalog number: SH30598.01 )
    12. HyCloneTM L-glutamine (GE Healthcare, HyCloneTM, catalog number: SH3003401 )
    13. HyCloneTM penicillin streptomycin 100x solution (GE Healthcare, HyCloneTM, catalog number: SV30010 )
    14. Gentamicin (50 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15750060 )
    15. Geneticin® selective antibiotic (G418 sulfate) (powder) (Thermo Fisher Scientific, GibcoTM, catalog number: 11811023 )
    16. Trypsin 0.05% protease solution with porcine trypsin, HBSS, EDTA; without calcium, magnesium (GE Healthcare, HyCloneTM, catalog number: SH30236.01 )
    17. ProFection® mammalian transfection system-calcium phosphate (Promega, catalog number: E1200 )
    18. Calcium chloride (CaCl2)
    19. 100 mM sodium pyruvate solution (GE Healthcare, HyCloneTM, catalog number: SH30239.01 )
    20. 1 M HEPES solution (GE Healthcare, HyCloneTM, catalog number: SH30237.01 )
    21. D10 medium (see Recipes)

  2. Differentiation of monocyte derived dendritic cells (MDDCs)
    1. 60 ml syringe with BD Luer-LokTM tip (BD, Luer-LokTM, catalog number: 309653 )
    2. Needle (50/sp, 500/ca), 18 G x 1 ½ in. (BD, SafetyGlideTM, catalog number: 305918 )
    3. 3 ml plastic disposable transfer (Pasteur) pipettes, sterile in 20s (Elkay Laboratory Products, Liquipette®, catalog number: 127-P503-20S )
    4. 40 μm nylon cell strainer
    5. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
    6. 10 cm plate (100 x 20 mm) (Corning, Falcon®, catalog number: 353003 )
    7. 50 ml buffy coat from New York Blood Center (leukapharesis)
    8. Ficoll-Paque PLUS (GE Healthcare, HyCloneTM, catalog number: 17-1440-02 )
    9. Dulbecco’s phosphate buffered saline (DPBS), without calcium, magnesium, phenol red (GE Healthcare, HyCloneTM, catalog number: SH30028.02 )
    10. Bovine serum albumin (BSA), fraction V, heat shock (Roche Diagnostics, catalog number: 03116956001 )
    11. EDTA
    12. RPMI 1640 media (GE Healthcare, HyCloneTM, catalog number: SH30096.01 )
    13. Fetal bovine serum (FBS)
    14. 1 M HEPES solution (GE Healthcare, HyCloneTM, catalog number: SH30237.01 )
    15. 2-mercaptoethanol (55 mM) (1,000x) (Thermo Fisher Scientific, GibcoTM, catalog number: 21985-023 )
    16. HyCloneTM L-glutamine (GE Healthcare, HyCloneTM, catalog number: SH3003401 )
    17. HyCloneTM penicillin streptomycin 100x solution (GE Healthcare, HyCloneTM, catalog number: SV30010 )
    18. Geneticin® selective antibiotic (G418 sulfate) (powder) (Thermo Fisher Scientific, GibcoTM, catalog number: 11811023)
    19. Anti-human CD14 microbeads
    20. 20.Human GM-CSF (Affymetrix, eBioscience, catalog number: 34-8339-82 )
    21. Human IL-4 (Affymetrix, eBioscience, catalog number: 34-8049-82 )
    22. Anti-hDC-SIGN PE (Clone: 120507), mouse IgG2B (R&D Systems, catalog number: FAB161P )
    23. Anti-hCD14 APC (Clone: 61D3) (Affymetrix, eBioscience, catalog number: 17-0149-42 )
    24. MACS buffer (see Recipes)
    25. DC medium (see Recipes)

  3. MDDC loading with HIV-1 or dextran
    1. 96 well clear round bottom TC-treated cell culture microplate, with lid, individually wrapped, sterile (Corning, Falcon®, catalog number: 353077 )
    2. X4-HIV-Gag-iGFP plasmid
      Note: Derived from HIV-1 molecular clone NL4-3 modified to insert the GFP protein in between the MA and CA domains of Gag as previously described (Hubner et al., 2007)
    3. Dextran, fluorescein (10,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-1820 )
    4. Dextran, fluorescein (70,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-1822 )
    5. Dextran, fluorescein (500,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-7136 )
    6. Dextran, fluorescein (2,000,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-7137 )
    7. Dulbecco’s phosphate buffered saline (DPBS), without calcium, magnesium, phenol red (GE Healthcare, HyCloneTM, catalog number: SH30028.02 )
    8. MACS buffer (see Recipes)

  4. Analysis of HIV-1 or dextran capture by flow cytometry
    1. Hanks’ balanced salt solution (HBSS) (1x) (Mediatech, catalog number: 21-023-CV )
    2. LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (for UV excitation) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L23105 )
    3. Anti-hDC-SIGN PE (Clone: 120507), mouse IgG2B (R&D Systems, catalog number: FAB161P )
    4. Anti-hCD14 APC (Clone: 61D3) (Affymetrix, eBioscience, catalog number: 17-0149-42 )
    5. Fixation/Permeabilization Solution Kit (RUO) (Fixation/Permeabilization solution 125 ml and BD Perm/WashTM buffer 100 ml) (BD, Cytofix/CytopermTM, catalog number: 554714 )
    6. Anti-P24 KC57-RD1 (Beckman Coulter, catalog number: 6604667 )

  5. Analysis of HIV-1 capture by Quantitative PCR (QPCR)
    1. LightCycler® 480 multiwell plate 96, white (Roche Diagnostics, catalog number: 04729692001 )
    2. LightCycler® 480 SYBR Green I Master (Roche Diagnostics, catalog number: 04887352001 )
    3. TRIzol® reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596-018 )
    4. SuperScript® III first-strand synthesis system (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-051 )
    5. primers:
      GFP forward: 5’-ACGTAAACGGCCACAAGTTC-3’
      GFP Reverse: 5’-AAGTCGTGCTGCTTCATGTG-3’
      GAPDH forward: 5’-CCCATCACCATCTTCCAGGAGCG-3’
      GAPDH Reverse: 5’-GAGATGATGACCCTTTTGGCTCC-3’

  6. Analysis of HIV-1 or dextran capture by confocal microscopy
    1. FisherbrandTM microscope slides (25 x 75 x 1.0 mm, double frosted precleaned) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-552-5 )
    2. Cover slip, round, 5 mm diameter, #1 0211 glass (Corning, Falcon®, catalog number: CLS-1763-005 )
    3. Poly-L-lysine solution (Sigma-Aldrich, catalog number: P8920-100ML )
    4. Paraformaldehyde, 16% solution, EM grade (Electron Microscopy Sciences, catalog number: 15710 )
    5. PIPES (1,4-piperazinediethanesulfonic acid) (Sigma-Aldrich, catalog number: P6757-100G )
    6. EGTA [Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid tetrasodium salt, ≥ 97%] (Sigma-Aldrich, catalog number: E8145-10G )
    7. Magnesium sulfate (MgSO4)
    8. Potassium hydroxide (KOH)
    9. TritonTM X-100 (Sigma-Aldrich, catalog number: X100-100ML )
    10. 5% casein solution as described and prepared in Dustin et al. (2007)
    11. 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
    12. 1,4-diazabicyclo[2.2.2]octane (DABCO) (Sigma-Aldrich, catalog number: D27802-25G )
    13. Poly(vinyl alcohol) (PVA) (Sigma-Aldrich, catalog number: 341584-25G )
    14. Glycerol (Sigma-Aldrich, catalog number: G5516-100ML )
    15. Tris buffer, pH 8.7 (1.5 M) (Bio-Rad Laboratories, catalog number: 1610798 )
    16. 2x PHEM buffer (see Recipes)
    17. DABCO-PVA (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5810 )
  2. Vortex
  3. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 240 )
  4. Flow cytometer (BD, model: LSRII )
  5. High throughput sampler (HTS) (BD)
  6. QPCR system (Roche Diagnostics, model: LightCycler 480 )
  7. Confocal microscope (Zeiss, model: LSM 710 )

Software

  1. ImageJ software

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Ménager, M. M. and Littman, D. R. (2016). Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs). Bio-protocol 6(22): e2004. DOI: 10.21769/BioProtoc.2004.

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分类

细胞生物学 > 细胞成像 > 荧光

免疫学 > 宿主防御 > 人

细胞生物学 > 基于细胞的分析方法 > 流式细胞术

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