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

doi:10.1038/protex.2018.064

Method Article

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

https://doi.org/10.1038/protex.2018.064

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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.

Biological techniques

flow cytometry

Mreg

miTreg

macrophage

T cell

IDO

Foxp3

Helios

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

See figure in Figures section.

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)

The authors declare no competing financial interest

supplement0.pdf
Table 1 List of antibodies

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

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