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
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posted 14 Sep, 2015
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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.
"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
recover the PBMC pellet in Baxter medium and count it at 1/100
Magnetic separation of Treg cells
Magnetic separation of Teff cells
Magnetic separation and preparation of APC (if needed)
Count the APC fraction
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)
incubate at 37°C during 20h to 72h
CFSE labelling of the Teff cells
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
distribute the different solutions : Teffs cells + beads (100 ul), Treg cells (50 ul) then TNF (50 ul) in the appropriate wells in duplicate for each condition.
Suppressive assay with APC and coated anti-CD3
distribute the different solutions : Teffs cells + APC (100 ul), Treg cells (50 ul) then TNF (50 ul) in the appropriate wells in duplicate for each condition.
Suppressive assay with APC and soluble anti-CD3
distribute the different solutions : Teffs cells + APC + anti-CD3 (100 ul), Treg cells (50 ul) then TNF (50 ul) in the appropriate wells in duplicate for each condition.
FACS Analysis
resuspend in 200 ul of PBS+3%FCS and analyse by FACS
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 14 Sep, 2015
You are reading this latest protocol version
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