This protocol describes step by step how to perform a suppressive assay with human Treg cells recovered from blood sample.
Method Article
Human Treg cell suppressive assays
https://doi.org/10.1038/protex.2015.078
This work is licensed under a CC BY-NC 3.0 License
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version
This protocol describes step by step how to perform a suppressive assay with human Treg cells recovered from blood sample.
In vitro Treg suppression assays are performed to determine the suppressive function of Treg cells on Teff cells. They are performed by co-culturing the responding population (Teff cells) with the Treg cells stimulated by APC or anti-CD3 coated beads.
Blood sample
anti-CD25 coated CliniMACS (Miltenyi Biotec)
anti-CD4 coated (Miltenyi Biotec)
anti-CD14 coated (Miltenyi Biotec)
anti-CD19 coated (Miltenyi Biotec)
LS columns (Miltenyi Biotec)
Baxter medium (PBS 1X supplemented with 5% of sodium citrate and 5% of human serum albumine)
Culture medium (RPMI-1650 medium (Gibco) supplemented with 10% heat-inactivated FCS (Gibco or Hyclone), 292 µg/ml or 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco))
IL-2 (Novartis)
TNF-α (PeproTech and R&D Systems)
U-bottom 96-well plates (Corning Costar, 3799)
48-well plates (Corning Costar, 3548)
CFSE (Sigma-Aldrich)
anti-CD3/CD28 beads (LifeTechnologies)
"List of antibodies":http://www.nature.com/protocolexchange/system/uploads/3807/original/Table1.?1438096118
MidiMACS separator
MACS Multistand
FACS Aria (BD biosciences)
FACS LSRII (BD biosciences)
PBMC isolation by Ficoll Hypaque gradient centrifugation
Magnetic separation of Treg cells
Magnetic separation of Teff cells
Magnetic separation and preparation of APC (if needed)
FACS sorting of Treg cells
anti-CD25 PE (1/20)
anti-CD127 BV451 (1/40)
anti-CD45RA PE-Cyanine7 (1/10)
CD4+CD25+IL-7Ra-CD45RA- for memory Treg cells
Treg cell preincubation (if needed)
CFSE labelling of the Teff cells
wash the Teff cells in PBS and centrifugate them at 300G during 5 minutes at 4°C
repeat the step 1.
incubate the Teff cells with 1 uM CFSE at 1 x 10^7 cells per ml in PBS during 5 minutes at RT
stop the reaction with 1/5 of FCS during 1 minute
wash in Complete medium and centrifugate the cells at 300G during 5 minutes at 4°C
repeat step 5.
Count the Teff cells
All suppressive assays are performed in U-bottom 96-well plates with 104 CFSE labelled Teff cells and 104 to 1.25 x 10^3 Treg cells (1:1 to 1:8) in 200 µl of culture medium at 37°C 5% CO2 during 4 days. Some wells with Teff cells alone are used as positive control of proliferation and are also used to determine the direct effect of TNF on Teff cells.
Suppressive assay with beads
Suppressive assay with APC and coated anti-CD3
Suppressive assay with APC and soluble anti-CD3
FACS Analysis
Calcul of the percentage of suppression
For each Teff/Treg ratio, the percentage of suppression was calculated with the following formula: [Log2(y) of (Teff cells alone) - Log2(y) of (Teff + Treg cells]) / Log2(y) of (Teff cells alone) X 100. The y value corresponds to the mean fluorescent intensity of CFSE of the whole Teff cell population divided by the mean fluorescent intensity of CFSE of undivided Teff cells.
The authors declare no competing financial interests.
Table1 List of antibodies
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version