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

Published: August 31, 2023

Abstract

Source: Lu, Y., et alIn Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) 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 (THGM). Isolated naïve T cells are stimulated for differentiation into THGM, 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 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. Prepare 50 mL of complete RPMI medi…

Offenlegungen

The authors have nothing to disclose.

Materials

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

Tags

Play Video

Diesen Artikel zitieren
An In Vitro Technique to Differentiate GM-CSF Producing T Helper Cells from Naïve T Cells. J. Vis. Exp. (Pending Publication), e21575, doi: (2023).

View Video