A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

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

1. Perform all steps under a 70% ethanol-cleaned biological hood.
2. 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.
3. Supplement the remaining 430 mL of RPMI 1640 1x with 50 ml inactivated fetal bovine serum (FBS), 5 mL penicillin/streptomycin, 5 mL N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 5 ml non-essential amino acids, 5 mL sodium pyruvate, and 0.05 m....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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