An In Vitro Technique to Differentiate GM-CSF Producing T Helper Cells from Naïve T Cells

Abstract

Source: Lu, Y., et al. In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (T_HGM) Cells. J. Vis. Exp. (2018).

This video demonstrates the in vitro differentiation of granulocyte-macrophage-colony-stimulating factor or GM-CSF-producing T helper cells (T_HGM). Isolated naïve T cells are stimulated for differentiation into T_HGM, which is confirmed by detecting high intracellular GM-CSF production and a low expression of other T helper cell-specific cytokines.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Reagent and Material Preparation

1. Prepare 500 mL of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA).
   NOTE: Keep the buffer on ice throughout the whole procedure for optimal results.
2. Prepare 50 mL of complete RPMI media containing RPMI 1640, 10% FBS, and 1% penicillin-streptomycin. Use complete RPMI media containing 50 µM β-mercaptoethanol for the T_H<....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Biowest	L0500-500
FBS Heat-Inactivated	Capricorn	FBS-HI-12A
Penicillin-Streptomycin	Gibco	15140122
10x PBS	1st Base	BUF-2040-10	Diluted to 1x PBS in sterile water
EDTA	1st Base	BUF-1053-100ml-pH8.0
10X ACK buffer	Biolegend	420301	diluted in sterile water
Cell strainer	SPL Life Sciences	93070
CD4 microbeads	Miltenyi Biotec	130-049-201
2-Mercaptoethanol	Sigma	M7522
LS column	Miltenyi Biotec	130-042-401
Magnetic stand	Miltenyi Biotec	130-042-303
1ml syringe	Terumo	SS+01T
Centrifuge	Eppendorf	Eppendorf 5810R
Sony SY3200 cell sorter	Sony	Sony SY3200 cell sorter
50ml conical centrifuge tube	Greiner Bio-One	210261
15m conical centrifuge tube	Greiner Bio-One	188271
FACS tube	Corning	352054
anti-mouse CD4 PerCP-eFluor 710	eBioscience	46-0041-82
PE-conjugated anti-mouse CD25 (IL-2Ra, p55)	eBioscience	12-0251-82
anti-human/mouse CD44 APC	eBioscience	17-0441-82
anti-mouse CD62L FITC	eBioscience	11-0621-85
Neubauer-improved counting chamber	Marienfeld	640010
Hyclone Trypan Blue Solution	GE healthcare Life Sciences	SV30084.01
Microscope	Nikon	Nikon Elipse TS100
Purified anti-mouse CD3e antibody	Biolegend	100314
Purified hamster anti-mouse CD28	BD Biosciences	553295
Purified Rat anti-mouse IFNγ	eBioscience	16-7312-85
Purified anti-mouse IL-4 Antibody	Biolegend	504102
Recombinant Mouse IL-7 Protein	R&D system	407-ML
PMA	Merck Millipore	19-144
Ionomycin	Sigma	I0634
GolgiPlug protein transport inhibitor	BD Biosciences	51-2301KZ
Intracellular Fixation&Permeabilization buffer set	eBioscience	88-8824-00
anti-mouse GM-CSF PE	eBioscience	12-7331-82
FITC anti-mouse IL-17A	Biolegend	506908
APC anti-mouse IFN-gamma	Biolegend	505810