In vivo DCs Depletion with Diphtheria Toxin and MARCO⁺/MOMA1⁺ Cells Depletion with Clodronate Liposomes in B6.CD11c-DTR Mice

Materials and Reagents

1. 25 gauge needle (BD Biosciences, catalog number: 305122 )
   
2. Cell strainer (100 μm pore size) (Corning Incorporated, catalog number: 352360 )
   
3. 15 ml tubes (TPP, catalog number: 91015 )
   
4. 1 ml syringes (BD Biosciences, catalog number: 309659 )
   
5. 1 ml syringes with 30 gauge needle (BD Biosciences, catalog number: 328278 )
   
6. B6 mice (Jackson Laboratories, model: B6 )
   
7. B6.CD11c-DTR mice (Jackson Laboratories, model: C57BL/6 )
   
8. Diphtheria toxin (Sigma-Aldrich, catalog number: D0564 )
   
9. Sodium clodronate (Melone Pharmaceutical, catalog number: 22560-50-5 )
   
10. RPMI 1640 (Thermo Fisher Scientific, catalog number: 11875093 )
    
11. Fetal bovine serum heat inactivated (Thermo Fisher Scientific, catalog number: 10437028 )
    
12. Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140122 )
    
13. L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
    
14. Sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360070 )
    
15. 2-mercaptoethanol Thermo Fisher Scientific, catalog number: 21985023 )
    
16. Halothane (Sigma-Aldrich, catalog number: H0150000 )
    
17. Monoclonal antibody (mAb) to MARCO (R&D Systems, catalog number: FAB2956P )
    
18. mAb to MOMA-1 (Abcam, catalog number: ab51814 )
    
19. mAb to F4/80 (eBioscience, catalog number: 47-4801 )
    
20. mAb to CD11b (BD Biosciences, catalog number: 562950 )
    
21. mAb to CD11c (eBiosciences, catalog number: 17011482 )
    
22. mAb to I-Ab (eBiosciences, catalog number: 46532082 )
    
23. NaCl
    
24. KCl
    
25. Na₂HPO₄
    
26. KH₂PO₄
    
27. 1x phosphate buffered saline (PBS) (see Recipes)
    
28. Staining Buffer (see Recipes)
    
29. Supplemented RPMI 1640 (see Recipes)
    
30. ClLip (see Recipes)

Equipment

1. Centrifuge (Eppendorf, model: 5804 )
   
2. Laminar flow hood (AirClean Systems, catalog number: AC8000HLF )
   
3. FACSCanto II Flow Cytometer, 8-color, lasers blue/red/violet (BD Biosciences, catalog number: 338962 )
   
4. Push-Pull syringe pump (KD Scientific, Model: KDS120 )
   

Procedure

1. DCs depletion in B6.CD11c-DTR mice
   1. Inject B6.CD11c-DTR mice (6-8 weeks age) i.p. with 2 ng/g of body weight of DTx (200 μl volume per mouse), or with PBS as depletion control (200 μl volume per mouse), using a syringe with 25 gauge needle.
      
   2. 24 h after injection, mice are euthanized with halothane by inhalation (or other approved euthanasia protocol).
      
   3. The spleens are removed and processed in a cell strainer inside a sterile culture hood (with 5 ml of supplemented RPMI 1640), followed by two washes with RPMI (at 300 x g, 5 min, 4 °C).
      
   4. Splenocytes are then stained with mAbs (0.5 µl per 10⁶ cells – each mAb at initial 0.5 mg/ml concentration, diluted in 25 µl of Staining Buffer) to CD11c and I-Ab (1 incubation period of 30 min), re-suspended in staining buffer (200 µl) and analyzed in a FACSCanto device, to evaluate depletion efficiency.
      
      
2. MZMΦs depletion in B6 mice with ClLip
   
   1. Inject B6 mice (6-8 weeks age) i.v. with 8.5 µg/g of body weight of ClLip (200 µl per mice) prepared as described in (van Rooijen et al., 1993), or with PBS-loaded liposomes (200 µl per mice, 8.5 µg/g of body weight) as depletion control, using a syringe with 30 gauge needle.
      
   2. 24 h to seven days after injection, mice are euthanized with halothane by inhalation (or other approved euthanasia protocol) and bled by cardiac puncture with a 1 ml syringe with a 25 gauge needle.
      Note: In both time points only MZMΦs are depleted in our protocol (this was done as a control to ensure only MZMΦs are depleted in our protocol, opposite to injection of higher concentrations of ClLip).
      
   3. The spleens are removed and processed in a cell strainer inside a sterile culture hood (with 5 ml of supplemented RPMI 1640), followed by two washes with RPMI (at 300 x g, 5 min, 4 °C).
      
   4. Splenocytes are then stained with mAbs (0.5 µl per 10⁶ cells – each mAb at initial 0.5 mg/ml concentration, diluted in 25 µl of Staining Buffer) to MARCO, MOMA-1, CD11b, CD11c and I-Ab (1 incubation period of 30 min), re-suspended in Staining Buffer (200 µl) and analyzed in a FACSCanto device, to evaluate depletion efficiency. An example of results obtained with this experiment is shown in Figure 1A and 1B, respectively.
      Note: Different DC subsets might have different sensitivity to DTx treatment, with different repopulation rates following initial depletion. Thus, for prolonged experiments, an important step would be to perform repopulation rate assays for the DCs subsets of interest.
      
      
      Figure 1. In vivo depletion of splenic DCs and MZMΦs. A. Representative plots of mice depleted of DCs using as controls of depletion B6 mice treated with DTX (here, CD11c-DTR mice treated with DTx present with a lower percentage of CD11c⁺I-A⁺ DCs, as showed in the gate in the upper right of each histogram (this decrease in percentage is an indicative of cell depletion). Adapted from Borges da Silva et al. (2015). B. Representative plots of mice depleted of MZMΦs using as controls B6 mice treated with ClLip (here, ClLip-treated mice present with a lower percentage of MARCO⁺MOMA-1⁺ MZMΦs, as showed in the gates represented in each histogram (this decrease in percentage is an indicative of cell depletion). Adapted from Borges da Silva et al. (2015).
      

Recipes

1. 1x phosphate buffered saline (PBS)
   Dissolve the following in 800 ml distilled H₂O
   8 g NaCl
   0.2 g KCl
   1.44 g Na₂HPO₄
   0.24 g KH₂PO₄
   Adjust pH to 7.4
   Adjust volume to 1 L with additional distilled H₂O
   Sterilize the solution
   Note: Adjust the pH using HCl and NaOH.
   
2. Staining Buffer
   Dissolve the following in 200 ml PBS
   1 ml 10% azide
   2 ml fetal bovine serum heat inactivated
   
3. Supplemented RPMI 1640
   Dissolve the following in 200 ml RPMI 1640 medium
   2 ml L-glutamine
   20 ml fetal bovine serum heat inactivated
   2 ml sodium pyruvate
   2 ml Penicillin-Streptomycin
   200 µl 2-mercaptoethanol
   
4. ClLip
   1. Inject (0.2 ml/min) an ethereal solution of 50 mg phosphatidylcholine and 8 mg cholesterol into 5 ml of a 50 mM/L clodronate aqueous solution maintained at 42 °C, by using a syringe adapted in a Push-Pull syringe pump, equipped with a fine-gauge needle (N^o 3D).
      
   2. During injection, a nitrogen stream will be infused into the clodronate solution, up to liposome formation and removal of residual solvent.
      
   3. Centrifuge liposome suspension (22,800 x g, 30 min, 25 °C). Wash twice with PBS, and resuspend in 2 ml PBS.
      
   4. Filter through a 0.8 Am polycarbonate membrane.     
      

Acknowledgments

HBdS was supported by an award from FAPESP (number: 2014/00810-5) and MRDL was supported by a grant from FAPESP (number: 2013/07140-2), and from CNPq 303676/2014-0 (MRDL) and 448765/2014-4 (MRDL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The DTX-mediated depletion of DCs was adapted from Jung et al. (2002), and the ClLip-mediated depletion of MZMΦs was adapted from McGaha et al. (2011).

References

1. Borges da Silva, H., Fonseca, R., Cassado Ados, A., Machado de Salles, E., de Menezes, M. N., Langhorne, J., Perez, K. R., Cuccovia, I. M., Ryffel, B., Barreto, V. M., Marinho, C. R., Boscardin, S. B., Alvarez, J. M., D'Imperio-Lima, M. R. and Tadokoro, C. E. (2015). In vivo approaches reveal a key role for DCs in CD4⁺ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria. PLoS Pathog 11(2): e1004598.
   
2. Jung, S., Unutmaz, D., Wong, P., Sano, G., De los Santos, K., Sparwasser, T., Wu, S., Vuthoori, S., Ko, K., Zavala, F., Pamer, E. G., Littman, D. R. and Lang, R. A. (2002). In vivo depletion of CD11c⁺ dendritic cells abrogates priming of CD8⁺ T cells by exogenous cell-associated antigens. Immunity 17(2): 211-220.
   
3. McGaha, T. L., Chen, Y., Ravishankar, B., van Rooijen, N. and Karlsson, M. C. (2011). Marginal zone macrophages suppress innate and adaptive immunity to apoptotic cells in the spleen. Blood 117(20): 5403-5412.
   
4. van Rooijen, N. and van Kesteren-Hendrikx, E. (2003). "In vivo" depletion of macrophages by liposome-mediated "suicide". Methods Enzymol 373: 3-16.