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