A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds
Published: November 30, 2023
Abstract
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.
Protocol
1. Preparation of Splenocyte Medium and Flow-cytometry Buffer Perform all steps under a 70% ethanol-cleaned biological hood. Remove 70 mL of pre-warmed Roswell Park Memorial Institute (RPMI) 1640 1x medium (commercially available and supplied in filtered 500 ml volume sterile bottles) and place in a sterile tube. The removed medium will be used later in the protocol. Supplement the remaining 430 mL of RPMI 1640 1x with 50 ml inactivated fetal bovine serum (FBS), 5 mL penic…
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The authors have nothing to disclose.
Materials
| 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 |
