This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Standard Protocols for Flow Cytometry for monocytes, macrophages, DC and T cells
Paloma Riquelme
Department of Surgery, University Hospital Regensburg
Corresponding Author
James A. Hutchinson
Department of Surgery, University Hospital Regensburg
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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.
Keywords
flow cytometry, Mreg, miTreg, macrophage, T cell, IDO, Foxp3, Helios
Figure 1
Figure 2
Figure 3
Figure 4
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
- Make stock solution of 10% NaN3 in DPBS
- Transfer 497ml DPBS to a Schott bottle
- Add 1 ml 10% NaN3 solution to DPBS for an end concentration of 0.02% NaN3
- Add 2 ml 0.5 M EDTA to give an end concentration of 2 mM
- Add 5 g BSA and allow to dissolve
Store solution at 4°C
2. PROTOCOLS FOR CELL-SURFACE STAINING OF MACROPHAGES, DC AND T CELLS
2.1 Standard extracellular staining protocol
- Harvest cells into cold DBPS. 2 × 105 cells are required for each staining reaction.
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 100 µl/reaction of FACS buffer + 10% FcR block
- Incubate at 4°C for 30 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl/tube
- Add primary antibodies as indicated
- Incubate at 4°C in dark for 20 min
- Add 5 µl 7-AAD per reaction. Incubate at 4°C in dark for 10 min
- Add 2 ml DBPS per tube. Vortex
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump.
Resuspend cells in 200 µl DPBS for analysis by flow cytometry.
2.2 Amplification of PE or APC signals using FASER kit
- Harvest cells into cold DBPS. 2 × 105 cells are required for each staining reaction.
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 100 µl / reaction of FACS buffer + 10% FcR block
- Incubate at 4°C for 30 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
- Add primary PE or APC conjugated antibody as indicated
- Incubate at 4°C in dark for 20 min
- Add 5 µl 7-AAD per reaction. Incubate at 4°C in dark for 10 min
- Add 2 ml DBPS per tube. Vortex
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump.
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 1
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 2
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 1
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 2
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
Resuspend cells in 200 µl DPBS for analysis by flow cytometry.
3. INTRACELLULAR STAINING OF HUMAN MREGS FOR IDO
- Harvest cells into cold DBPS. 5 × 105 Mregs are required for each staining reaction
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 3 ml DBPS
- Add 0.75 µl Fixable Live-Dead dye
- Incubate at 4°C in dark for 30 min
- Wash one time in 10 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 300 µl FACS buffer + 10% FcR block for 15 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
- Add primary PE-conjugated CD33 or isotype antibody as indicated in Figure 1 (3 reactions)
- Incubate at 4°C in dark for 30 min
- Wash samples one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Add 100 µl IC Fixation Buffer for 30 min RT in the dark
- Wash samples twice in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
- Resuspend samples in 100 µl of FACS buffer + 10% FcR block for 15 min at 4°C
- Add intracellular staining antibodies as indicated for 30 min at RT in the dark
- Wash samples once in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
Resuspend cells in 200 µl DPBS for analysis by flow cytometry
See figure in Figures section.
4. INTRACELLULAR STAINING OF HUMAN TREGS FOR FOXP3 AND HELIOS
4.1 Intracellular staining of cultured T cells
- Harvest cells into cold DBPS. 5 × 105 cells are required for each staining reaction
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 1 ml DBPS
- Add 1 µl Fixable Live-Dead dye
- Incubate at 4°C in dark for 30 min
- Wash one time in 10 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 100 µl FACS buffer + 10% FcR block for 15 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
- Add cell surface antibodies as indicated in Figure 2
- Incubate at 4°C in dark for 30 min
- Wash samples one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump. Gentle vortex
- Add 500 µl Fixation/Permeabilization
- Incubate at 4°C in dark for 30 min
- Wash samples in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
- Add intracellular staining antibodies (FoxP3-PE, optional Helios-APC) as indicated
- Incubate for 30 min at 4°C in the dark
- Wash samples 3 times in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
Resuspend cells in 200 µl Perm Buffer for analysis by flow cytometry
See figure in Figures section.
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
See figure in Figures section.
5. Gating strategy for identifying TIGIT+ Tregs among NSG mouse splenocytes
- Hutchinson J.A. et al. MITAP-compliant characterization of human regulatory macrophages. Transpl Int. 30(8):765-775 (2017).
- 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)
- Hutchinson J.A. et al. Human regulatory macrophages. Methods Mol Biol. 677:181-92 (2011)
This is a list of supplementary files associated with this preprint. Click to download.
- Listofantibodies.pdf
Table 1 List of antibodies
Version History
CURRENT STATUS: Posted
Version 1
Posted 11 Jul, 2018
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
Method Article
Standard Protocols for Flow Cytometry for monocytes, macrophages, DC and T cells
Paloma Riquelme
Department of Surgery, University Hospital Regensburg
Corresponding Author
James A. Hutchinson
Department of Surgery, University Hospital Regensburg
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 11 Jul, 2018
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
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
- Make stock solution of 10% NaN3 in DPBS
- Transfer 497ml DPBS to a Schott bottle
- Add 1 ml 10% NaN3 solution to DPBS for an end concentration of 0.02% NaN3
- Add 2 ml 0.5 M EDTA to give an end concentration of 2 mM
- Add 5 g BSA and allow to dissolve
Store solution at 4°C
2. PROTOCOLS FOR CELL-SURFACE STAINING OF MACROPHAGES, DC AND T CELLS
2.1 Standard extracellular staining protocol
- Harvest cells into cold DBPS. 2 × 105 cells are required for each staining reaction.
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 100 µl/reaction of FACS buffer + 10% FcR block
- Incubate at 4°C for 30 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl/tube
- Add primary antibodies as indicated
- Incubate at 4°C in dark for 20 min
- Add 5 µl 7-AAD per reaction. Incubate at 4°C in dark for 10 min
- Add 2 ml DBPS per tube. Vortex
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump.
Resuspend cells in 200 µl DPBS for analysis by flow cytometry.
2.2 Amplification of PE or APC signals using FASER kit
- Harvest cells into cold DBPS. 2 × 105 cells are required for each staining reaction.
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 100 µl / reaction of FACS buffer + 10% FcR block
- Incubate at 4°C for 30 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
- Add primary PE or APC conjugated antibody as indicated
- Incubate at 4°C in dark for 20 min
- Add 5 µl 7-AAD per reaction. Incubate at 4°C in dark for 10 min
- Add 2 ml DBPS per tube. Vortex
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump.
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 1
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 2
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 1
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 80 µl FACS buffer + 20 µl FcR block + 10 µl FASER reagent 2
- Incubate at 4°C in dark for 10 min
- Wash one time in 2 ml DPBS by centrifugation at 300 g for 6 min
Resuspend cells in 200 µl DPBS for analysis by flow cytometry.
3. INTRACELLULAR STAINING OF HUMAN MREGS FOR IDO
- Harvest cells into cold DBPS. 5 × 105 Mregs are required for each staining reaction
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 3 ml DBPS
- Add 0.75 µl Fixable Live-Dead dye
- Incubate at 4°C in dark for 30 min
- Wash one time in 10 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 300 µl FACS buffer + 10% FcR block for 15 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
- Add primary PE-conjugated CD33 or isotype antibody as indicated in Figure 1 (3 reactions)
- Incubate at 4°C in dark for 30 min
- Wash samples one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Add 100 µl IC Fixation Buffer for 30 min RT in the dark
- Wash samples twice in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
- Resuspend samples in 100 µl of FACS buffer + 10% FcR block for 15 min at 4°C
- Add intracellular staining antibodies as indicated for 30 min at RT in the dark
- Wash samples once in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
Resuspend cells in 200 µl DPBS for analysis by flow cytometry
See figure in Figures section.
4. INTRACELLULAR STAINING OF HUMAN TREGS FOR FOXP3 AND HELIOS
4.1 Intracellular staining of cultured T cells
- Harvest cells into cold DBPS. 5 × 105 cells are required for each staining reaction
- Pellet cells by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump
- Resuspend cells in 1 ml DBPS
- Add 1 µl Fixable Live-Dead dye
- Incubate at 4°C in dark for 30 min
- Wash one time in 10 ml DPBS by centrifugation at 300 g for 6 min
- Resuspend cells in 100 µl FACS buffer + 10% FcR block for 15 min
- Transfer blocked cell suspensions into FACS tubes at 100 µl / tube
- Add cell surface antibodies as indicated in Figure 2
- Incubate at 4°C in dark for 30 min
- Wash samples one time in 2 ml DPBS by centrifugation at 300 g for 6 min
- Aspirate supernatant using vacuum pump. Gentle vortex
- Add 500 µl Fixation/Permeabilization
- Incubate at 4°C in dark for 30 min
- Wash samples in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
- Add intracellular staining antibodies (FoxP3-PE, optional Helios-APC) as indicated
- Incubate for 30 min at 4°C in the dark
- Wash samples 3 times in 2 ml Perm Buffer by centrifugation at 300 g for 6 min
Resuspend cells in 200 µl Perm Buffer for analysis by flow cytometry
See figure in Figures section.
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
See figure in Figures section.
5. Gating strategy for identifying TIGIT+ Tregs among NSG mouse splenocytes
- Hutchinson J.A. et al. MITAP-compliant characterization of human regulatory macrophages. Transpl Int. 30(8):765-775 (2017).
- 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)
- Hutchinson J.A. et al. Human regulatory macrophages. Methods Mol Biol. 677:181-92 (2011)