Protocol Exchange

Author Information

Subject Terms

Keywords

Abstract

Introduction & Materials

Procedures

Outcomes

Conflicting Financial Interests

Associated Publications

Supplementary Files

Figures

Paloma Riquelme

James A. Hutchinson

Figure 1

Figure 2

Figure 3

Figure 4

Standard Protocols for Flow Cytometry for monocytes, macrophages, DC and T cells

This document describes standard research protocols used for analysis of marker expression by flow cytometry in the Hutchinson work-group. In Riquelme et al., these protocols were applied to the analysis of monocyte-derived macrophages and DC, and cultured T cells.

**General reagents**





Biocoll Separating Solution \(L6115, Millipore-Biochrom)


DPBS without Ca2+ or Mg2+ \(D8537, Sigma)


BSA \(01400.1, Biomol)


NaN3 \(S8032, Sigma)


EDTA 0.5M \(15575-038, Gibco Life)


Human FcR blocking reagent \(130-059-901, Miltenyi)


7-AAD \(559925, BD)


Intracellular fixation buffer \(00-8222, eBiosciences)


Permeabilization Buffer \(00-8333, eBiosciences)


FOXP3 Fixation/Permeabilization \(00-5521-00, eBiosciences)


Fixable Live-Dead Viability DyeFluor660 \(65-0864, eBiosciences)


Fixable Live-Dead Viability DyeFluor506 \(65-0866, eBiosciences)


Faser Kit APC \(130-091-762, Miltenyi)





**Antibodies**


Please see Table 1

FACSCanto II cytometer: Standard configuration with 8 colour, 3 lasers \(BD Biosciences)


DivaTM Software version v.6.1.3 \(BD Biosciences)


Navios cytometer: Standard configuration with 10 colours, 3 lasers \(Beckman Coulter)


Navios clinical software \(Beckman Coulter)


FlowJoTM analysis software v.7.6.5 \(FlowJo, LLC.)


Kaluza analysis software v.1.0 \(Beckman Coulter)

**1. REAGENT PREPARATION** 





**1.1 Preparation of FACS buffer**


1. Make stock solution of 10% NaN3 in DPBS
 2. Transfer 497ml DPBS to a Schott bottle 
 3. Add 1 ml 10% NaN3 solution to DPBS for an end concentration of 0.02% NaN3
 4. Add 2 ml 0.5 M EDTA to give an end concentration of 2 mM
 5. Add 5 g BSA and allow to dissolve
 6. Store solution at 4°C
 





**2. PROTOCOLS FOR CELL-SURFACE STAINING OF MACROPHAGES, DC AND T CELLS**





**2.1 Standard extracellular staining protocol**


1. Harvest cells into cold DBPS. 2 × 105 cells are required for each staining reaction.
 2. Pellet cells by centrifugation at 300 g for 6 min
 3. Aspirate supernatant using vacuum pump
 4. Resuspend cells in 100 µl/reaction of FACS buffer + 10% FcR block
 5. Incubate at 4°C for 30 min
 6. Transfer blocked cell suspensions into FACS tubes at 100 µl/tube
 7. Add primary antibodies as indicated
 8. Incubate at 4°C in dark for 20 min
 9. Add 5 µl 7-AAD per reaction. Incubate at 4°C in dark for 10 min
 10. Add 2 ml DBPS per tube. Vortex
 11. Pellet cells by centrifugation at 300 g for 6 min
 12. Aspirate supernatant using vacuum pump.
 13. Resuspend cells in 200 µl DPBS for analysis by flow cytometry.
 


**2.2 Amplification of PE or APC signals using FASER kit**


1. Harvest cells into cold DBPS. 2 × 105 cells are required for each staining reaction.
 2. Pellet cells by centrifugation at 300 g for 6 min
 3. Aspirate supernatant using vacuum pump
 4. Resuspend cells in 100 µl / reaction of FACS buffer + 10% FcR block
 5. Incubate at 4°C for 30 min
 6. Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
 7. Add primary PE or APC conjugated antibody as indicated
 8. Incubate at 4°C in dark for 20 min
 9. Add 5 µl 7-AAD per reaction. Incubate at 4°C in dark for 10 min
 10. Add 2 ml DBPS per tube. Vortex
 11. Pellet cells by centrifugation at 300 g for 6 min
 12. Aspirate supernatant using vacuum pump.
 13. Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 1
 14. Incubate at 4°C in dark for 10 min
 15. Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
 16. Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 2
 17. Incubate at 4°C in dark for 10 min
 18. Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
 19. Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 1
 20. Incubate at 4°C in dark for 10 min
 21. Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
 22. Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 2
 23. Incubate at 4°C in dark for 10 min
 24. Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
 25. Resuspend cells in 200 µl DPBS for analysis by flow cytometry.
 





**3. INTRACELLULAR STAINING OF HUMAN MREGS FOR IDO**





1. Harvest cells into cold DBPS. 5 × 105 Mregs are required for each staining reaction
 2. Pellet cells by centrifugation at 300 g for 6 min
 3. Aspirate supernatant using vacuum pump
 4. Resuspend cells in 3 ml DBPS 
 5. Add 0.75 µl Fixable Live-Dead dye
 6. Incubate at 4°C in dark for 30 min
 7. Wash one time in 10 ml DPBS by centrifugation at 300 g for 6 min
 8. Resuspend cells in 300 µl FACS buffer + 10% FcR block for 15 min
 9. Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
 10. Add primary PE-conjugated CD33 or isotype antibody as indicated in Figure 1 \(3 reactions)
 11. Incubate at 4°C in dark for 30 min
 12. Wash samples one time in 2 ml DPBS by centrifugation at 300 g for 6 min
 13. Add 100 µl IC Fixation Buffer for 30 min RT in the dark
 14. Wash samples twice in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
 15. Resuspend samples in 100 µl of FACS buffer + 10% FcR block for 15 min at 4°C
 16. Add intracellular staining antibodies as indicated for 30 min at RT in the dark
 17. Wash samples once in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
 18. Resuspend cells in 200 µl DPBS for analysis by flow cytometry
 


<a href="#figures" data-url="http://www.nature.com/protocolexchange/system/uploads/6619/original/Figures_Flow_Fig_2.jpg?1523899046">See figure in Figures section.</a>








**4. INTRACELLULAR STAINING OF HUMAN TREGS FOR FOXP3 AND HELIOS**





**4.1 Intracellular staining of cultured T cells**


1. Harvest cells into cold DBPS. 5 × 105 cells are required for each staining reaction
 2. Pellet cells by centrifugation at 300 g for 6 min
 3. Aspirate supernatant using vacuum pump
 4. Resuspend cells in 1 ml DBPS 
 5. Add 1 µl Fixable Live-Dead dye
 6. Incubate at 4°C in dark for 30 min
 7. Wash one time in 10 ml DPBS by centrifugation at 300 g for 6 min
 8. Resuspend cells in 100 µl FACS buffer + 10% FcR block for 15 min
 9. Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
 10. Add cell surface antibodies as indicated in Figure 2
 11. Incubate at 4°C in dark for 30 min
 12. Wash samples one time in 2 ml DPBS by centrifugation at 300 g for 6 min
 13. Aspirate supernatant using vacuum pump. Gentle vortex
 14. Add 500 µl Fixation/Permeabilization
 15. Incubate at 4°C in dark for 30 min
 16. Wash samples in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
 17. Add intracellular staining antibodies \(FoxP3-PE, optional Helios-APC) as indicated
 18. Incubate for 30 min at 4°C in the dark
 19. Wash samples 3 times in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
 20. Resuspend cells in 200 µl Perm Buffer for analysis by flow cytometry
 


<a href="#figures" data-url="http://www.nature.com/protocolexchange/system/uploads/6621/original/Figures_Flow_cytometry_Fig_3.jpg?1523899091">See figure in Figures section.</a>





**4.2 Intracellular staining for FoxP3 in T cells from clinical blood samples**





1 1 ml blood is used for each staining reaction. Dilute blood 1:1 with DBPS


2 Layer blood carefully onto 6 ml Biocoll in a 15 ml tube


3 Centrifuge at 2000 rpm, 20°C, for 25 min with no brake


4 Recover cell layer in the interface


5 Wash with 14 ml DPSB by centrifugation at 1600 rpm for 10 min


6 Aspirate supernatant using vacuum pump


7 Resuspend cells in 100 µl / reaction of FACS buffer + 10% FcR block


8 Incubate at 4°C for 30 min


9 Transfer blocked cell suspensions into FACS tubes at 100 µl / tube


10 Add cell surface antibodies as indicated in Figure 3


11 Incubate at 4°C in dark for 30 min


12 Add 2 ml DBPS per tube. Vortex


13 Pellet cells by centrifugation at 300 g for 6 min


14 Aspirate supernatant using vacuum pump. Gentle vortex


15 Add 500 µl Fixation/Permeabilization


16 Incubate at 4°C in dark for 30 min


17 Wash samples in 2 ml Perm Buffer by centrifugation at 300 g for 6 min


18 Add intracellular staining antibodies as indicated


19 Incubate for 30 min at RT in the dark


20 Wash samples 3 times in 2 ml Perm Buffer by centrifugation at 300 g for 6 min


21 Resuspend cells in 200 µl Perm Buffer for analysis by flow cytometry





<a href="#figures" data-url="http://www.nature.com/protocolexchange/system/uploads/6623/original/Figures_Flow_cytometry_FOXP3_Tigit_small.jpg?1523900494">See figure in Figures section.</a> 








**5. Gating strategy for identifying TIGIT+ Tregs among NSG mouse splenocytes**





<a href="#figures" data-url="http://www.nature.com/protocolexchange/system/uploads/6625/original/Figures_Flow_cytometry_NSG_small.jpg?1523900496">See figure in Figures section.</a>

1. Hutchinson J.A. et al. MITAP-compliant characterization of
  human regulatory macrophages. Transpl Int. 30\(8):765-775 \(2017).





2. Hutchinson J.A. et al. Cutting Edge: Immunological consequences and
  trafficking of human regulatory macrophages administered to renal transplant


recipients. J Immunol. 1;187\(5):2072-8 \(2011)





3. Hutchinson J.A. et al. Human regulatory
  macrophages. Methods Mol Biol. 677:181-92 \(2011)

CC BY 4.0

This document describes standard research protocols used for analysis of marker expression by flow cytometry in the Hutchinson work-group. In Riquelme et al., these protocols have been applied to the analysis of monocyte-derived macrophages, DC and cultured T cells.

Method Article

Paloma Riquelme, James A. Hutchinson

Paloma Riquelme

Department of Surgery, University Hospital Regensburg

Corresponding Author

James A. Hutchinson

Department of Surgery, University Hospital Regensburg

Corresponding Author

DOI:

10.1038/protex.2018.064

Download PDF

License:

This work is licensed under a CC BY 4.0 License. Read Full License

Abstract

Keywords

flow cytometry, Mreg, miTreg, macrophage, T cell, IDO, Foxp3, Helios

Figures

Introduction

Reagents

Equipment

Procedure

References

Competing Interests

Supplementary Files

Comments

Version History

CURRENT STATUS: Posted

Version 1

No community comments so far

Metrics

Comments: 0

HTML Views: ...

Subject Areas

Biological techniques

Method Article

Paloma Riquelme, James A. Hutchinson

Paloma Riquelme

Department of Surgery, University Hospital Regensburg

Corresponding Author

James A. Hutchinson

Department of Surgery, University Hospital Regensburg

Corresponding Author

Badges

Peer Review Timeline

Version History

CURRENT STATUS: Posted

Version 1

No community comments so far

Metrics

Metrics

Comments: 0

HTML Views: ...

Subject Areas

Subject Areas

Biological techniques

DOI & PDF

DOI:

10.1038/protex.2018.064

Download PDF

License

License:

This work is licensed under a CC BY 4.0 License. Read Full License

Declarations

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

References

Comments

Related Articles

Privacy Policy

Terms of Service