使用Celltrace Violet示踪法进行体外T淋巴细胞增殖和调节性T细胞抑制实验

Kristofor K. Ellestad; Colin C. Anderson

doi:10.21769/BioProtoc.1698

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Peer-reviewed

Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet

使用Celltrace Violet示踪法进行体外T淋巴细胞增殖和调节性T细胞抑制实验

KE Kristofor K. Ellestad email

CA Colin C. Anderson email

发布: 2016年01月05日第6卷第1期 DOI: 10.21769/BioProtoc.1698 浏览次数: 32282

评审: Ivan ZanoniAndrea PuharAnonymous reviewer(s)

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Cover of European Journal of Immunology, featuring study using the protocol.

Dec 2014

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Abstract

Measurement of the incorporation of radionuclides such as ³H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately quantitate T cell divisions, and an inability to combine proliferation analyses with simultaneous evaluation of the expression of cellular markers in divided cells. By labeling T cells with reactive dyes such as CFSE, Celltrace Violet, and others that are partitioned equally between daughter cells at each cell division, one can relatively easily track generations of proliferated cells and their expression of various molecules by flow cytometry.

FoxP3⁺ regulatory T cells (Treg) are critical mediators of immune tolerance and evaluation of their functionality is an important step in characterizing many immune models (Rudensky, 2011). Classically CD4⁺ Treg and conventional or “responder” T cells have been isolated based on their surface expression of CD25 (Treg: CD4⁺CD25⁺, Tresp: CD4⁺CD25^-). However, we and others have noted that populations of CD4⁺CD25^- cells express the FoxP3 transcription factor and have suppressive function. Therefore we have utilized the transgenic FoxP3-EGFP mouse to facilitate live purification of suppressor and responder populations based on EGFP (and thus FoxP3) expression. Here we present our adapted protocol for assaying regulatory T cell suppression of Celltrace Violet-labeled responder T cells.

Keywords: Treg (调节性T细胞)

Proliferation (增殖)

Suppression (抑制)

Regulatory t cell (调节 T 细胞)

Flow cytometry (流量细胞计数法)

Materials and Reagents

70 μm nylon mesh cell strainers (Thermo Fisher Scientific, Fisherbrand^TM, catalog number: 22363548 )
5 ml polypropylene round-bottom FACS tubes (BD Falcon^®, catalog number: 352063 )
Note: Currently, it is “Corning, catalog number: 352063”.
50 m conical polypropylene tubes (BD Falcon^®, catalog number: 352098 )
Note: Currently, it is “Corning, catalog number: 352098”.
96 well plate, U bottom (BD, Falcon^®, catalog number: 353077 )
Note: Currently, it is “Corning, catalog number: 353077”.
Mice
1. B6-FoxP3^EGFP strain (B6.Cg-Foxp3^tm2Tch/J) (Jackson Laboratories, catalog number: 006772 ) (see Note 1)
2. Rag KO (Rag 1 KO, B6.129S7-Rag^1tm1Mom/J) (Jackson Laboratories, catalog number: 002216 )
Biosure preservative-free 8x sheath fluid concentrate (for cell sorter) [Cedarlane, catalog number: 1027(BS) ]
Hank’s Balanced Salt solution (HBSS) (Life Technologies, Invitrogen^TM, catalog number: 14170 )
Note: Currently, it is “Thermo Fisher Scientific, catalog number: 14170”.
Fetal bovine serum (Life Technologies, Invitrogen^TM, catalog number: 16170078 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 16170078”.
1 M HEPES (Life Technologies, Invitrogen^TM, catalog number: 15630080 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 15630080”.
PE anti-mouse CD8a (clone 53-6.7) (eBioscience, catalog number: 120081 )
APC anti-mouse CD4 (clone GK1.5) (eBioscience, catalog number: 170041 )
PerCP-eFluor 710 anti-mouse CD4 (clone RM4-5) (eBioscience, catalog number: 460042 )
APC-eFluor 780 anti-mouse TCRβ (clone H57-597) (eBioscience, catalog number: 475961 )
Ultracomp eBeads (eBioscience, catalog number: 012222 )
“Fc blocking” antibody, anti-mouse CD16/CD32 (clone 2.4g2) (9.6 mg/ml) (Bio X cell, catalog number: CUS-HB-197 )
Functional grade purified anti-mouse CD3ε (clone 145-2C11) (eBioscience, catalog number: 160031 )
Celltrace violet cell proliferation kit (Life Technologies, Invitrogen^TM, catalog number: C34557 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: 16170078”.
LIVE/DEAD Fixable Yellow Dead cell stain kit (Life Technologies, Invitrogen^TM, catalog number: L34959 ) or 7-Aminoactinomycin D (7-AAD) (Sigma-Aldrich, catalog number: A9400)
Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: L34959”.
18 MΩ^.cm deionized water
Dulbecco modified Eagle medium (DMEM) (Life Technologies, Invitrogen^TM, catalog number: 11965 )
Note: Currently, it is “Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11965 ”.
Pen/strep (100 U/ml Penicillin, 100 μg/ml Streptomycin) (Life Technologies, Invitrogen^TM, catalog number: 15140122 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122”.
Non-essential amino acids (Life Technologies, Invitrogen^TM, catalog number: 11140050 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 11140050”.
Sodium pyruvate (Life Technologies, Invitrogen^TM, catalog number: 11360070 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 11360070”.
L-glutamine (Life Technologies, Invitrogen^TM, catalog number: 25030081 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081”.
2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Enriched DMEM (E-DMEM) (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)
ACK lysis buffer (see Recipes)

Equipment

Cell sorter (BD Biosciences, model: BD Influx )
Flow cytometer with 405 nm excitation capability (see note 3) (BD Biosciences, model: BD LSR II )
37 °C humidified incubator, 5% CO₂
Centrifuge (capable of spinning 50 ml conical tubes, 5 ml FACS tubes and 96 well plates)
Multichannel pipette (30-300 μl) (Eppendorf)

Procedure

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Ellestad, K. K. and Anderson, C. C. (2016). Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet. Bio-protocol 6(1): e1698. DOI: 10.21769/BioProtoc.1698.