Source: Fouda, A., et al., A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules. J. Vis. Exp., (2017).
This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds' stimulatory and inhibitory effects, respectively.
1. Preparation of Splenocyte Medium and Flow-cytometry Buffer
2. Generation of Splenocyte Cell Suspension from Nur77GFP Mouse Spleens
3. T-cell Isolation from the Splenocyte Cell Suspension
4. T-cell Activation and the Induction of green fluorescent protein (GFP) Expression
5. High Throughput Screening (HTS) of Small Molecules
The authors have nothing to disclose.
Nur77GFP mice | The Jackson Laboratory | Mouse strain No. 016617 | An in house colony was established at our animal facility |
96 wells-U culture plates, sterile | VWR International | 10062-902 | T-cell activation using the magnetic beads |
70µm cell strainer, sterile | Corning Inc. | 352350 | Generation of splenocytes cell suspension |
5 ml polystyrene round bottom tubes, sterile | Corning Inc. | 352058 | Generation of splenocytes cell suspension |
50 ml polypropylene conical bottom tubes, sterile | VWR International | 89039-656 | Generation of splenocytes cell suspension |
10 ml syringe without needle, sterile | Becton, Dickinson and Company | 305482 | To mash the spleen |
5 ml cell culture dish, sterile | Greiner Bio-One | 627 160 | To mash the spleen |
Penicillin- Streptomycin (10,000 U/mL) | WISENT Inc. | 450-200-EL | Component of the splenocyte media |
RPMI 1600 with sodium bicarbonate and L- glutamine | WISENT Inc. | 350-002-CL | Component of the splenocyte media |
MEM non-essential amino acids | WISENT Inc. | 321-010-EL | Component of the splenocyte media |
HEPES free acid 1 M | WISENT Inc. | 330-050-EL | Component of the splenocyte media |
Sodium pyruvate solution (100mM) | WISENT Inc. | 600-110-EL | Component of the splenocyte media |
Fetal Bovine Serum (FBS) | WISENT Inc. | 080-910 | Component of the splenocyte media and flow-cytometry buffer |
Phosphate buffered saline (PBS) | WISENT Inc. | 311-010-CL | Component of flow-cytometry buffer |
2-Mercaptoethanol (55mM) | Thermo Fisher Scientific | 21985-023 | Component of the splenocyte media |
T- Cells isolation kit | Stemcell Technologies | 19851 | To isolate T cells |
Mouse T-Activator CD3/CD28 superparamagnetic beads | Thermo Fisher Scientific | 11452D | To activate T cells |
Cell isolation magnet | Stemcell Technologies | 18000 | To isolate T cells and remove the magnetic beads |
Recombinant Murine IL-7 | Peprotech | 217-17 | To support T-cell survival during activation |
Anti-mouse CD3 antibody | BD Pharmingen | 561799 | To stain T cells for flow-cytometry |
Biomed FXp | PerkinElmer Inc. | A31842 | To re-suspend cells after 24 hours incubation |
Opera Phenix High Content Screening System | PerkinElmer Inc. | HH14000000 | To analyze GFP/Hoechst signal |